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Appendix 1
Practical Tips for Two-Photon Microscopy
Mark B. Cannell, Angus McMorland, and Christian Soeller
INTRODUCTION
As is clear from a number of the chapters in this volume, 2-photon
microscopy offers many advantages, especially for living-cell
studies of thick specimens such as brain slices and embryos.
However, these advantages must be balanced against the fact that
commercial multiphoton instrumentation is much more costly than
the equipment used for confocal or widefield/deconvolution. Given
these two facts, it is not surprising that, to an extent much greater
than is true of confocal, many researchers have decided to add a
femtosecond (fs) pulsed near-IR laser to a scanner and a microscope to make their own system (Soeller and Cannell, 1996; Tsai
et al., 2002; Potter, 2005). Even those who purchase a commercial
multiphoton system find that it helps to understand a bit more about
how to optimize the performance of the fs laser system.1 This
Appendix has been added to the Handbook to provide the basic
alignment and operating information that such people need.
First, the safety announcement . . .
LASER SAFETY
Light sources for multiphoton microscopy are almost without
exception very powerful pulsed lasers (laser class IV). It is vital
that any personnel who perform alignment or other operations that
carry a risk of beam exposure are familiar with and follow laser
safety regulations. During routine operation one MUST ensure
that accidental exposure to the pulsed laser beam is prevented by
providing proper shielding and interlocks.
During alignment, protective eyewear is not an option — it is
essential!
See
http://www.osha.gov/SLTC/laserhazards/
for
US
guidelines.
blue and green diode lasers. To provide an alignment beam to
which the external laser can be aligned, light from this reference
laser needs to be bounced back through the microscope optical
train and out through the external coupling port:
CAUTION: Before you switch on the reference laser in this
configuration make sure that all PMTs are protected and/or
turned off.
Place a front-surface mirror on the stage of the microscope and
focus onto the reflective surface using an air objective for convenience (at sharp focus, you should be able to see scratches or other
mirror defects through the eyepieces). The idea of this method is
to cause the reference laser beam to bounce back through the
optical train and emerge from the other laser port. To do this, select
filter settings that will allow some of the light from the internal
laser to exit the chosen coupling port. In order to bring two laser
beams to co-linearity, a beam-steering device is essential. A singlemirror beam steerer provides angular control while changing the
separation between the mirrors of a 2-mirror steerer provides beam
translation (Fig. A1.1).
It is also possible to achieve beam translation with a second
angular control mirror. After adjusting the incoming near-IR beam
to an intensity where it can be viewed without totally overwhelming the reference beam,2 adjust one mirror to make both laser spots
merge at the surface of the other (angle-adjustable) mirror. Then
that mirror is adjusted to bring the beams to co-linearity. We find
it useful to use a piece of light-blue paper as this shows the dimmed
infrared beam well. If the laser has been tuned to the far part of
the spectrum, you may have to use an IR viewer or viewer card to
visualize the beam.
TESTING ALIGNMENT AND
SYSTEM PERFORMANCE
Just as in any other type of microscopy, correct optical alignment
is crucial for achieving optimal, diffraction-limited performance in
2-photon microscopy. The alignment of external lasers such as the
Ti : S or similar 2-photon sources into a laser scanning microscope
can be simplified if a well-aligned “internal” or reference laser is
available. In commercial confocal microscopes, typical candidate
lasers include Argon-ion or green HeNe lasers or, more recently,
On a regular basis and particularly subsequent to laser alignment,
the performance of the multiphoton microscope should be tested.
The prime indicator of proper alignment of an imaging system is
its point-spread function, as measured by using a sample containing sub-resolution fluorescent beads. A test slide can be prepared
by letting a drop of diluted beads dry onto a coverslip. The beads
are then embedded in a drop of Sylgard elastomer (Dow Corning,
USA) with a microscope slide placed on top. We usually use
0.2 mm beads from Molecular Probes (Eugene, OR). These are
available in a range of colors suitable for 2-photon microscopy. It
1
2
LASER ALIGNMENT
The Multiphoton Users Group e-mail network at ·mplsm-users@
yahoogroups.comÒ, operated by Steve Potter at Georgia Tech, enrolled its
500th member in 2003.
As is explained below, this can be achieved by over-closing the slit and/or
reducing pump power, because mode-locking is not required. We typically
use <20 mw @ 800 nm and <10 mW at 720 nm.
Mark B. Cannell, Angus McMorland, and Christian Soeller • Department of Physiology, FMHS, University of Auckland, New Zealand
900
Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006.
Practical Tips for Two-Photon Microscopy • Appendix 1
A
B
901
C
FIGURE A1.1. (A) 2D simplification of the beam alignment process using a conventional beam-steerer. A vertical translation of a tilted mirror is used to bring
the two beams to a common point on a second, tiltable mirror. (B) Rotation of the second mirror at the point of the common spot makes the two beams co-linear.
(C) The co-linear beams after alignment.
takes only about 30 minutes to prepare 10 slides in this way. Once
the elastomer has set, these slides will last for months if kept in a
dark drawer. As a result, they provide a good standard to check the
microscope sensitivity and resolution provided you have recorded
microscope and laser settings (including center wavelength, laser
power and bandwidth/pulse length) with each reference image.
With proper alignment, the beads should blur approximately
evenly as you focus above and below them. Asymmetric blurring
above-and-below best focus indicates spherical aberration while
motion of the centroid of intensity means that the objective
aperture is filled asymmetrically. The spatial resolution (without a
pinhole) should be similar to confocal performance, values
between 0.2–0.4 mm in plane, full-width at half maximum
(FWHM) and 0.5–0.8 mm out of plane (in the z direction) should
be attainable when using a high-numerical-aperture (NA ~1.3)
objective.
A very weak and noisy signal can have a number of causes. If
there is no problem with the detectors or emission filters (most of
which would also be apparent when operating the microscope with
conventional [1-photon] laser excitation), check that the laser
beam fills the objective rear aperture fully and evenly by rotating
the objective turret to an empty position, placing a lens tissue over
the opening and inspecting the pattern of illumination (using an IR
viewer if necessary). The beam should be accurately centered in
the empty socket and should form a uniform circle of light that
will cover the rear aperture (~8–10 mm wide) of a typical objective lens. If the light intensity at the rear aperture is low (<10 mW)
make sure that no IR-opaque optical items are obstructing the illumination path.3 It is also possible that the beam is so badly misaligned that only scattered light is being observed. You can check
for this by ensuring that adjustments of the alignment mirrors have
the expected effects on the spot in the BFP.
If the microscope is a combined confocal/multiphoton system,
the bead slide is also a useful tool to disclose alignment offsets
between the 2-photon laser system and any other lasers. In particular you should check for any axial offsets (i.e., focus shifts), par-
3
If little light is coming out of the objective, it may be the anti-reflection coatings that are at fault. Coatings used to reduce reflection losses in the visible
may become mirrors in the near-IR. See the transmission tables in Chapter 7
and its Appendix.
ticularly if the system is to be used for 2-photon flash photolysis
or combined confocal and multiphoton co-localization studies.
In our laboratory we perform a basic system test with a prepared bead sample on a daily basis. This check (usually conducted
following system startup) is well worth the ~5 minutes it takes,
especially if it helps avoid debugging signal problems later when
a precious biological sample is on the stage.
LASER SETTINGS AND OPERATION
Historically, the mode-locked lasers used for 2-photon imaging
could be quite temperamental and ensuring that proper laser operation was a large part of the challenge of running a multiphoton
microscope. With the advent of fully computer-controlled turn-key
laser systems, this has become less of an issue. In any case, as the
most versatile source for 2-photon imaging is still the tunable
Ti : S laser in the femtosecond configuration, we will focus on it
here. Regardless of whether you are using a fully automated or a
manually adjusted Ti : S system, it is important to monitor and
optimize the laser output before imaging.
The choice of center wavelength is generally determined by
the fluorochromes to be excited. As a general rule of thumb you
should try to use the longest wavelength compatible with the dyes
in your sample as this will help minimize photodamage and also
reduce scattering of the excitation light. Data on excitation spectra
is now available from many sources in the literature and, if in
doubt, there are mailing lists where one can ask other researchers
for advice (see http://groups.yahoo.com/group/mplsm-users/ and
http://listserv.acsu.buffalo.edu/archives/confocal.html).
MONITORING LASER PERFORMANCE
During tuning and imaging, laser operation can be very conveniently monitored using a spectrum analyzer. We use a system
made by Rees Instruments (currently available models include the
Rees E200 series laser spectrum analyzers by Imaging and Sensing
Technology Ltd., Alton, UK) to monitor a secondary beam containing only a small fraction of the total output power. During laser
tuning, this device allows one to measure the center wavelength
and, more importantly, the width of the spectrum. The spectral
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Appendix 1 • M.B. Cannell et al.
width of the beam, as displayed by the analyzer, provides the feedback for optimizing the slit width and position to obtain modelocked operation (with manually tuned laser systems). The start of
mode-locked operation is indicated by the change of the spectral
shape from one or a small number of sharply defined lines which
indicate continuous wave (CW) operation, see Figure A1.2A, to an
A
B
C
D
E
G
approximately Gaussian-shaped output spectrum which may have
a spike (Fig. A1.2B) indicating CW breakthrough. Optimal closure
of the slit leads to a smooth Gaussian-like spectrum (Fig. A1.2C)
which, in this case, is ~5nm wide (FWHM). At 750 nm this spectral width implies a 120 fs pulse. Closing the slit further can lead
to an oscillation of pulse amplitude (Q-switching), which is shown
F
FIGURE A1.2. (A–F) show spectrum analyzer output
during Ti : S tuning. The small gradations at the bottom
indicate 1nm. (A) Before mode-locking, the spectrum
consists of a few narrow spikes. (B) With mode-locking
underway, the spectrum increases in width, but the spike
indicates CW breakthrough. To cure this, the slit needs
to be closed more. (C) Optimal operation, the slit has
been closed just enough to stop CW but at the same time
not so closed that Q-switching starts, a mode of behavior shown in (D,E). To stop Q switching, more prism
must be inserted into the beam path (which will increase
system bandwidth) and/or the slit needs to be opened
(or even a reduction in pump power). (F) shows the shortest pulse that can be readily achieved with our Coherent
MIRA 900F system. The FWHM of the spectrum is
~14 nm at a center wavelength of ~750 nm. (G) shows the
relationship between the FWHM of the spectrum and the
pulse width for a transform-limited sec2 pulse, with
center wavelength indicated next to each curve. In this
case, a 14nm bandwidth from our laser (F) implies a
very short pulse width of 40 fs. Generally, we use
longer pulses (~120fs e.g., C) than this in imaging
experiments.
Practical Tips for Two-Photon Microscopy • Appendix 1
in the spectrum as oscillations (Figs. A1.2D, A1.2E) and should be
removed by re-opening the slit or increasing the intra-cavity group
velocity dispersion by moving the intra-cavity prism further in. By
suitable adjustment of the slit and the intra-cavity group velocity
dispersion, the pulse may be shortened and this will be reflected
in an increase the width of the output spectrum (Fig. A1.2F).
With our laser, a 14 nm FWHM bandwidth can be achieved
corresponding to a ~40 fs pulse at 750 nm. During imaging, Qswitching manifests itself as a sudden increase in image noise due
to aliasing between laser excitation and the pixel clock. A quick
look at the spectrum should indicate if the laser needs tuning to
remove this source of image noise.
Typically, pulses leaving a commercial Ti : S laser, as used for
2-photon microscopy, are ~100 fs long. Pulse length is an important variable that is most accurately determined with an optical
autocorrelator. However, from a practical point of view, a spectrum analyzer is easier to use than an autocorrelator and gives sufficient information on laser performance. The length of the laser
pulse is inversely proportional to the spectral FWHM during
mode-locked operation. Figure A1.2G shows this relationship for
various center wavelengths.
POWER LEVELS AND TROUBLE-SHOOTING
In our experience illumination power levels at the sample should
be kept <20 mW in living cells to minimize the risk of cell damage,
although that figure is dependent on the nature of the experiment,
the 2-photon absorber, the objective NA, and sample scattering.
Problems with mode-locked lasers in 3D microscopic imaging
most often arise from:
1. Pump laser noise (amplitude noise or beam-pointing
instability).
2. Pump laser alignment.
3. Dirt on mirrors.
4. Poor alignment within the cavity.
5. Stray reflections from surfaces that reflect energy back into
the cavity.
6. Poorly trained personnel changing the alignment between the
pump and the prisms of the Ti : S cavity over time.
7. UFM (unidentified fingerprints on mirrors!).
8. Air currents that affect beam-pointing stability.
9. Loss of alignment of laser to microscope.
10. Poor matching of laser beam profile to microscope aperture.
To address problems 1–4, the manufacturer generally provides
troubleshooting advice that should be consulted. Problem 5 can be
avoided by using an optical isolator, i.e., a device which allows
light to pass only in the forward direction but blocks back reflections. A simpler workaround (that has worked well in our hands)
involves slightly tilting strongly reflecting surfaces (e.g., neutral
density filters — see below) with respect to the optical axis. For
laser safety you should provide an appropriate beam dump for any
strong reflections off the optical axis. Problems 6 and 7 should be
resolved by the system manager. Problem 8 can be reduced by surrounding all beams with plastic tubes. Problem 9 can be ascertained using a reference laser, especially a laser built into the
microscope itself. Problem 10 arises from the laser beam being too
small to fully fill the objective rear aperture (so a loss of resolution occurs) or too large, in which case there is a loss of intensity
at the sample. In both cases, the problem can be fixed using laser
beam expansion (or compression) with a telescope (Galilean beam
expander). In our microscope we use ~4x expansion of the Ti : S
903
laser beam as a reasonable compromise between filling the real
aperture adequately and throughput. (We built a simple expander
from a plano-convex and a plano-concave lens which were singlelayer antireflection coated.) In addition, by focusing the beam
expander carefully, it is possible to minimize the axial shift of focal
plane between visible light and the IR.
CHOICE OF PULSE LENGTH
The dispersion of the pulse by the microscope optics is typically
>2000 fs2 at 800 nm. This suggests that the shortest pulse width that
can be delivered to the sample would be >100 fs unless group
velocity dispersion compensation is performed to “prechirp” the
pulse (Soeller and Cannell, 1996). Shorter pulses increase the ratio
of 3- to 2-photon excitation and, since 3-photon excitation at
800 nm would correspond to hard UV, such excitation is generally
undesirable. We therefore suggest that for routine operation
~120 fs pulses are probably optimal. Perhaps paradoxically, in the
absence of GVD compensation, a shorter pulse at the laser is translated to a much longer pulse at the sample. As it is hard to run a
conventional Ti : S laser with pulses longer than ~150 fs, longer
pulses at the sample may be produced by making very short pulses
(e.g., 40 fs) at the laser. See Chapters 5 and 28 for further discussion on pulse broadening.
CONTROLLING LASER POWER
Being able to control laser power electronically is useful because
it permits rapid suppression of the beam at the end of each scan
line where the beam slows and stops before retracing its path. This
slow movement subjects the parts of the specimen at either side of
the raster to very high integrated excitation which is very damaging. Unfortunately, the acousto-optic modulators (AOM), which
are commonly used for this purpose in visible light microscopes,
are less suitable for 2-photon because heating and birefringent
effects in the crystal reduce beam intensity stability. The squarelaw dependence of 2-photon excitation on input power amplifies
this instability at the sample. Additionally, because the modelocked laser beam has significant bandwidth (compared to a CW
laser) the beam will be dispersed if it is diffracted in the AOM. As
the rear aperture of the objective must be overfilled, this dispersion results in a loss of bandwidth and therefore a longer and misshapen pulse. This effect can be avoided if one uses the zero-order
(i.e., undiffracted) beam of the AOM for microscopy and the first
order beam is used simply to extract energy from it. However, as
only about 75% of the beam can be diffracted out, this approach
only reduces the beam to 25% of the input power.
A better alternative is to use a Pockels cell. While more expensive, these devices are much faster and more controllable than an
AOM, but they also suffer from some problems:
1. The Pockels cell has a limited lifetime that is dependent on the
time spent in the energized state.
2. Alignment is critical: the full power of the beam must pass
cleanly through the free aperture and not touch the interior of
the cell under any circumstance or damage will result.
3. High voltages are present.
It should be noted that, for ~120 fs pulses, dispersive broadening
by the Pockels is generally small and should therefore be of no
concern when it is used in a 2-photon imaging setup.
If rapid beam modulation is not needed, laser power can be
controlled by neutral density filters or a polarizer. Such neutral
904
Appendix 1 • M.B. Cannell et al.
density filters need to be of the reflecting type as high powers
destroy absorbing filters. The beam reflected from the filter needs
to be absorbed by something for safety and we use a “beam dump”
made of black anodized aluminum with a machined recess so it is
hard to see the dumped beam. Since the output beam of the laser
is polarized, beam intensity may be modulated by rotating a polarizer in front of it. Glass Glan-Thompson polarizers can be used but
plastic polarizers are quite unsuitable for typical power levels as
they melt (see also Attenuation of Laser beams in Chapter 5, this
volume.)!
AM I SEEING TWO-PHOTON EXCITED
FLUORESCENCE OR . . .
Sometimes it is unclear if a detected signal is due to multiphotonexcited fluorescence or if it is due to optical bleed-through of the
(much more intense) near-IR excitation light. Such bleed-through
can occur, for example, if one uses filters with an unknown
response in the near-IR region. A simple test to distinguish between
these possibilities can be made by taking a control image with the
multiphoton laser source running in CW mode (at similar power).
When using a mode-locked Ti : S laser with manually operated slit
this can easily be achieved by over-closing the slit until modelocking is lost and then reopening the slit with the starter mechanism disabled. If the signal in question disappears when using CW
illumination, it must be due to some sort of multiphoton excitation
(2- or 3-photon fluorescence, or second- or third-harmonic generation). However, this simple test does not replace the more complex
illumination-power vs. signal-intensity measurements needed to
fully characterize each of these high-order excitation processes.
STRAY LIGHT AND NON-DESCANNED
DETECTION
One of the attractions of 2-photon microscopy resides in the
improved penetration depth obtained when imaging in strongly
scattering biological samples such as brain slices (see Soeller and
Cannell, 1999). Central to this advantage is the need to collect
emitted photons that are also scattered and so may not be focused
by the microscope optics and are therefore lost at intermediate
apertures. This problem can be overcome by using a photomultiplier tube that is mounted close to the sample (so that the emitted
light does not pass through the scanning system) to create a “nondescanned detector.” Such detectors are arranged so that any
photons of the right color, regardless of where they originate, are
directed onto the photocathode. As a result, non-descanned
detection is also far more likely to pick up stray light from the
microscope surroundings than conventional confocal optics. For
example, in a normal laboratory, light from computer screens and
equipment LEDs can cause a strong background signal even when
the room lights are turned off. To shield your setup from this stray
light, you may need to fabricate suitable shields around the sample
from black material. Alternatively, you may shield the whole
microscope from the surroundings by enclosing it in a completely
light tight box. This can be conveniently combined with electrical
shielding by providing a Faraday cage around 3 sides and the top
of the instrument and fully closing it during imaging by drawing
a black curtain or blind across the fourth side. For safety reasons,
this cage and any blinds or curtain should be made from fire-proof
materials.
LASER POWER ADJUSTMENT FOR IMAGING
AT DEPTH
Although 2-photon excitation penetrates deeper into scattering
samples (such as brain), the loss of peak excitation power at the
focus caused by scattering and spherical aberration still leads to a
loss of signal at depth. The solution to this problem is to alter the
illumination power as a function of depth and this is where the
intensity modulation provided by the Pockels cell may be used
to advantage. There are alternative ways to achieve changes in
illumination power but all assume that the maximum power
available from the 2-photon laser is higher than is needed for
normal operation. Thus, a wheel of reflective neutral density (ND)
filters may be placed in the beam path, providing intensity control
to quantized levels appropriate for different imaging depths.
A second option is to use a continuously variable reflective
neutral density filter, which allows more precise control over laser
power, but requires either manual rotation during imaging or a
motorized filter wheel. We suggest that the ideal solution is to
automatically attenuate the laser beam. using a Pockels cell
supplied with a varying drive voltage controlled by the focus
position.
In our experience, the laser power needs to increase (roughly)
exponentially with depth (e.g., see Fig. A1.4 in Soeller and
Cannell, 1999) but the exponential factor is highly dependent on
the sample. Thus a control experiment may be needed where a
similar sample is labeled with fluorescent beads (~2 mm in diameter). For brain slices, or other tissues which can be perfused, this
can be achieved by injecting the beads into a blood vessel before
slicing. By imaging the beads at different depths, the depth dependence of the excitation may be determined and used in subsequent
experiments. (Using beads will give more reliable results than
simply staining the entire specimen with a dye as this avoids problems arising from non-uniform staining.) It is important to note
that not all the signal loss is due to reduced excitation as emitted
light is also lost by scattering and adsorption. Thus, even if complete compensation of signal loss with depth can be achieved by
raising excitation power, it is better to err on the side of caution as
delivering too much power into the preparation at any depth may
lead to other concerns — for example, heating and other higherorder effects (see Chapter 38, this volume). If 100 mW were delivered and (eventually) absorbed within 10mm3 of tissue, the
average rate of rise of temperature would be 2.5oC/s. Although this
power is close to the maximum that may be achieved by typical
2-photon microscopes, it is clearly in the range where heating
effects could become a serious problem.
SIMULTANEOUS IMAGING OF
MULTIPLE LABELS
Another advantage of 2-photon excitation is that the 2-photon excitation spectra of fluorochromes are wider than their 1-photon counterparts. Multiple labels may therefore be imaged simultaneously
by using a single excitation wavelength and multiple detectors with
appropriate optics to isolate each different emitted wavelength.
This approach has several benefits: (1) Removal of offset problems
caused by non-confocality of different lasers; (2) reduction in
imaging time (which may be important for imaging of live-cell
processes); (3) reduction in the total amount of laser exposure to
the tissue and (4) avoidance of chromatic aberrations. Care must
be taken to ensure that bleed-through from one channel to another
Practical Tips for Two-Photon Microscopy • Appendix 1
is minimized by the use of the optimal beam-splitters (see also
Chapter 3, this volume). If some bleed-through is unavoidable then
an accurate measurement of the amount of bleed-through can be
made by imaging, in all channels, control slides that contain the
individual fluorochromes. From these measurements, contributions from bleed-through from one channel to another can be estimated and removed by subtraction during post-imaging analysis
(so-called “spectral unmixing”).
MINIMIZE EXPOSURE DURING ORIENTATION
AND PARAMETER SETTING
In most applications, imaging parameters need to be established
by trial and observation prior to the commencement of image
acquisition. Common examples are scanning across tissue looking
for “that cover image” and then establishing the upper and lower
limits of a volume of interest. While the use of 2-photon excitation prevents photobleaching above and below the focal plane, inplane photobleaching can be severe and care must be taken to
avoid over-exposure of samples to illumination light during these
adjustment procedures.
The key is to think before imaging. For example, if the sample
needs to be located in focus, is full power really necessary or will
the detection of just a few photons be sufficient? It follows that
during setup, the detector gain should always be set high and laser
power as low as possible. Single scans should be used in preference to continuous scanning. Can the sample be moved to an unexposed region once the acquisition parameters are set? Once the
correct settings have been determined, then laser power can be
increased for actual imaging and focal-plane bleaching indicates
that the maximum amount of information available has been
extracted from the dye in the sample.
ULTRAVIOLET-EXCITED FLUOROCHROMES
The use of ultraviolet (UV) excited dyes in 1-photon imaging is
restricted by the opacity of conventional optical components at UV
wavelengths as well as by chromatic aberrations and by the cost
905
and size of UV lasers. 2-photon excitation of UV dyes does not
suffer from these problems because the excitation wavelengths are
near-infrared, in a range that is compatible with normal optics.
The ability to use UV dyes allows more labels, and colors, to be
used in multiple-labeling experiments. In addition, combining a
UV-excited probe emitting in the blue part of the spectrum
allows greater spectral separation from a yellow-red label. UV
dyes, in general, may be excited by 2-photons at wavelengths £750
nm. For example, the AlexaFluor 350 fluorochromes (Molecular
Probes, Eugene, OR) come in a range of forms. The nearUV-excited nucleic-acid probes DAPI and Hoechst are often so
well excited using 2-photon illumination that it is necessary to
use very low concentrations to prevent bleed-through into other
channels.
ACKNOWLEDGEMENTS
We would like to thank Tim Murphy (University of British
Columbia, Vancouver, CA) for helpful comments on the
manuscript.
REFERENCES
Potter, S.M., 2005, Two-photon microscopy for 4D imaging of living neurons.
In: Imaging in Neuroscience and Development. A Laboratory Manual, (R.
Yuste, and A. Konnerth, eds.), pp. 59–70, Cold Spring Harbor Laboratory
Press.
Soeller, C., and Cannell, M.B., 1996, Construction of a two-photon microscope
and optimisation of illumination pulse width. Pflugers. Archiv. 432:
555–561.
Soeller, C., and Cannell, M.B., 1999, Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc.
Res. Tech. 47:182–195.
Taal, P.S., Nishimura, N., Yoder, E.J., White, A., Doluick, E., and Kleinfeld,
D., 2002, Principles, design and construction of a two-photon scanning
microscope for in vitro and in vivo studies, In: Method for in vivo Optical
Imaging, (R. Frostig, ed), CRC Press, pp. 113–171.
Appendix 2
Light Paths of the Current Commercial Confocal Light
Microscopes Used in Biology
James B. Pawley
INTRODUCTION
Since biologists became aware of the confocal microscope in the
late 1980s, numerous optical designs have been introduced by
manufacturers to try to meet the often-contradictory requirements
of the biological microscopist. Although many of these designs are
discussed at greater length in other chapters of the Handbook, it
was thought that it might be both useful to the reader, and fairer
to those designs not discussed elsewhere, to provide the reader
with a concise compilation of all the designs now available.
To that end I requested optical diagrams and tabular information from all of the major suppliers of the instruments used by biologists for 3D microscopy1 and the items that they provided make
up the bulk of this Appendix. Often manufacturers were hesitant
to provide specific information about details such as PMTs or scanning speeds etc., because they realized that there was a good
1
We have neglected to include any information on the systems for widefield/
deconvolution only because the optical paths of such systems are fairly
straightforward, and not in need of explanation.
chance that such data would go out of date with their next product
announcement. However, I tried to apply the same criteria to all
the contributors and this is as good a place as any to thank the manufacturers for their splendid cooperation.
To assist the reader, some of the optical information considered most relevant to the optical performance of these instruments
has been collected in Table A2.1. Although such a table cannot
contain all of the relevant information about such complex instruments, the headings have been chosen to reflect those specifications indicated to be of prime importance in the other chapters of
the Handbook. Abbreviations are explained in the footnote.
Of course, the manufacturers are correct about this information going out of date. Fortunately the WWW is now there to bring
you up to date. Even when the models are all different, we hope
that the you find the column headings in the table of optical parameters useful as the basis of questions you might ask about future
models.
There has been no effort to compare the computer operating
systems used to control these instruments. I wish to emphasize that
this is not because I think such details unimportant, but rather
because software systems tend to change with great speed and,
in addition, operating systems are probably best assessed in person.
James B. Pawley • University of Wisconsin, Madison, Wisconsin, 53706
906
Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006.
Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2
907
FIGURE A2.1. Schematic of the BD-CARV II
light path. The variable intensity light from a
Hg/metal halide light source passes through an excitation filter before being defleted by a dichroic mirror
towards the sample. The excitation light passes
through a Nipkow spinning-disk containing multiple
sets of spirally-arranged pinholes placed in the
intermediate-image plane of the objective lens. The
column of excitation light is projected through 1000
pinholes to simultaneously scan the entire field once
every millisecond, thereby creating a full image of
the focal plane in real-time. The emitted light passes
through the dichroic mirror and the emission filter
before either entering the CCD camera or the binocular eye-piece. The pinhole disk can be moved in and
out of the light path to produce a confocal or a
widefield fluorescence image. A variable slit at the
image plane can be used to selectively illuminate an
area of the sample allowing Fluorescence Recovery
After Photobleaching (FRAP) to be performed. All
movable parts including the filter wheels, spinningdisk shutters, and mirrors are automated and are controlled via touchpad or third-party software. Figure
kindly provided by BD-Biosciences, (Rockville,
MS).
FIGURE A2.2. Schematic of the LaVision-BioTec TriM-Scope light path. Multifocal multiphoton microscopy using a beamsplitter built with flat optics. Light
from a fs, near-IR, pulsed laser first passes a polarizing attenuator and a beam-expander before entering a pre-chirp compensator. It is then formed into as many
as 64 beams of equal intensity and spacing by being reflected from an array of sliding, planar, optical elements. The linear array of beams is then deflected by
2, closely-spaced galvanometer mirrors and fed into the microscope by being reflected off a high-pass beam-splitter. Two-photon-excited fluorescence from any
dye located at the focus plane of the objective passes through the short-pass dichroic, and barrier filters to a CCD camera or other photodetector.2 Because of
the large number of parallel beams and the high-QE of the CCD camera, it is possible to obtain useful, optical-section images at up to 3.5 k frames/second and,
because the system relies on 2-photon excitation, bleaching is restricted to the focal plane. For more discussion see Chapter 29, this volume.
2
T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, (2001), High efficiency beam splitter for multifocal multiphoton microscopy, J. Microsc., 201:368–376.
908
Appendix 2 • J.B. Pawley
Table A2.1. Optical Parameters of Current Commercial Confocal Microscopes
Company
Model
Lasers/Arc
Retrace
protect/Laser
atten
Fiber
optics
Pre-optics
Beam
expander
Scanner
Fastest
line scan,
Hz
Pixel
times
Scanned
field,
diam. int
im. plane
Largest
Raster
Pixel
BDBiosciences
CARV II
X-Cite 120
Hg/halide
arc, 8-place
filter wheel
NA/intensity
controlled by
aperture
Liquidfilled light
guide
NA
NA
Single-sided
Petran disk
1 k fps
5 k rpm
~1 ms
21 mm
CCD
LavisionBiotech
TriMScope2
Ti-sapph
750–100 nm,
100 fs pulse
NA/
Attenuator
0.1–100%
Laser is
coupled
directly
NA
Yes
1–64 beams
scanned by 2
galvos
3.5 k fps
> 500 ns
20 mm,
adapted to
CCD used
CCD
Leica
TCS SP2 AOBS
Many,
351–633 nm
Yes/3
AOTFs
SM-PP
Laser-merge
Adjustable
Rotatable k-scan
1.4 k or
2.8 k in
bi-direct
>500 ns
22 mm
4096 ¥ 4096
MP RS
Ti-Sapph
Yes, EOM
SM-PP
Laser-merge
Adjustable
Rotatable k-scan
4 k, or 8k >500 ns
in bidirect
22 mm
4096 ¥ 4096
C1-plus
Up to 3,
408–638 nm
AOM (opt)
SM-PP
Laser-merge
Fixed
2 close galvos
500, 1k
in bidirect
>1.68us
@512 ¥
512
17 mm
2048 ¥ 2048
C1si
Up to 3,
408–638 nm
AOM
SM-PP
laser input
fiber. MM
emission
fiber
Laser-merge
Fixed
2 close galvos
500, 1k
in bidirect
From
17 mm
4.08us
w/scan
at
rotation
512 ¥ 512
in
spectral
mode
Up to
512 ¥
512 in
spectral
mode
FV 300
Many,
405–633 nm
IR port
Many,
active
stabilizer,
351–633 nm,
IR port
Hg arc
Yes, AOTF
SM-PP
Laser-merge
Fixed
2 close galvos
1 k or 2 k
bi-direct
>2 ms
20 mm
2048 ¥ 2048
Yes, AOTF
SM-PP
Laser-merge
Fixed
2 k or 4 k
bi-direct
>2 ms
18 mm
4096 ¥ 4096
NA/intensity
controlled by
aperture
NA
NA
NA
2 pair of close
galvos, separate
image/bleach
scanners,
circular bleach
Interchangeable
single-sided slitpattern disk,
3 k rpm
3 k rpm,
15 fps
>1 ms
18 mm
CCD
Single galvo
scans an array
of point sources
1 galvo, 1 AOD
2 kHz
2 ms
17
CCD
50 kHz
20–125 ns
Nikon
Olympus
FV 1000
DSU
Visitech
Yokogawa5
Zeiss
1
VT-infinity
Many,
405–647 njm
Yes, AOTF
SM-PP
Laser-merge
(opt)
NA
VT-Eye
Many,
351–647 nm
Yes, AOTF
SM-PP
Laser-merge
(opt)
Fixed
CSU 10
2 or 3 lines6
NA/AOTF
SM
3.5 mm
core
Laser-merge
Fixed
Double Petran
Disk w/microlenses
1800 rpm, ~1 ms
360 fps
13 ¥ 9.5 mm CCD
CSU 22
3 or 4 laser
lines
NA/AOTF
SM
3.5 mm
core
Laser-merge
Fixed
Double Petran
Disk w/microlenses
Variable,
to 5 k
rpm, 1 k
fps
~1 ms
13 ¥ 9.5 mm CCD
LSM510META
Many,
351–633 nm
(Ti : Sapph)
Yes, AOTF
0.05–100%
(AOM)
SM-PP
Laser-merge
Adjustable
2 close galvos
1.3 k or
2.6 k in
bi-direct
640 ns–
2.3 ms
18 mm
2048 ¥
2048
LSM 5 Pascal
Many,
405–633 nm
SM-PP
Laser-merge
Adjustable
2 close galvos
1.3 k or
2.6 k in
bi-direct
640 ns–
2.3 ms
18 mm
2048 ¥ 2048
LSM5-LIVE
Many,
405–635 nm
No/
Mechanical
attenuator
0.05–100%
Yes, AOTF
0.05–100%
SM-PP
Laser-merge
Adjustable
Cylindrical
1 galvo
line-scan
(>60 k)
16 ms–
120 fps,
20 ms
512 ¥ 512,
1010 fps,
512 ¥ 50
18 mm
1024 ¥ 1024
1024 ¥ 1024
Record transmitted light through disk.
As the TriMScope is actually a multi-focus multiphoton fluorescence illuminator with widefield detection onto a CCD, its performance depends a great deal on the performance of this device.
3
These numbers assume that the tube mag is 1¥.
2
Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2
909
Table A2.1. (Continued)
Zoom
range
Tube
mag
Beam
dump
NA
1.1¥
NA
5-place
dichroic
wheel
Selfaligning
ROI for
CCD mode
1¥
NA
Short-pass
dichroic
NA
(multiphoton
excited
only)
32 : 1
Yes
AcoustoOptic
Preset
32 : 1
Yes
Acoustooptic
Yes
Dichroic,
changes
with cube
infinite
3.8
infinite
Beamsplitter
Pinhole
alignment
Pinhole range
Spectral
selection
Photodetector
Channels
Reflected/
transmitted
Digitizer
z-motion
8-place filter
wheel
CCD or EMCCD
2-camera port
No/yes1
CCD
Piezo
±100 nm
(opt)
8-place filter
wheel,
spectrometer
(opt)
CCD or EMCCD, 1–32
PMT array
3-camera port
Yes/no
CCD,
(opt ion,
PMT,
12-bit)
Stepper
motor,
(peizo
opt)
Common
pinhole, adjust
20–800 mm
Prism,
motorized
mirrors
8
Yes/yes
12-bit
Galvo,
±40 nm
Fixed
Common
pinhole, adjust
20–800 mm
Prism,
motorized
mirrors
4 PMT,
cooling
option,
APD option
4 PMT,
cooling
option, +2
nondescanned
8
Yes/yes
12-bit
Galvo,
±40 nm
Fixed
w/focusable,
alignable
pinhole lens
Fixed
w/focusable,
alignable
pinhole lens
Common
pinhole,
30, 60, 100,
150 mm3
Common
pinhole,
30, 60, 100,
150 mm4
Replaceable
filter cubes
3 side-window
fiber-coupled
PMTs
4
Yes/yes
12-bit
Stepper,
±50 nm
3 diffraction
gratings for
2.5 nm, 5 nm,
and 10 nm
channel width
32 element
multianode
PMT
32 acquired
simultaneously
Yes/yes
12-bit
Stepper,
50 nm
increments
Dichroic,
cubes, 2
positions
Dichroic
wheel, 6
positions
Common
pinhole,
alignable
Common
pinhole,
alignable
5 sizes
Dichroic filter
cube
3 PMTs, 2
fluor, 1 trans
3, 2 fl, 1 trans
Yes/yes
12-bit
Stepper,
±10 nm
adjust
50–800 or
50–300 on
spectral
2 diff-grating
channels,
motorized
slits
5 PMTs, 2
spectral, 1
trans, Photon
counting mode
5, 4 fl, 1 trans
Yes/yes
12-bit
Stepper,
±10 nm
Dichroic,
changes
with cube
Fixed 70 mm,
180 m spacing
10 : 1
(infinity)
3.42¥
Yes
50 : 1
(infinity)
3.82¥
Yes
NA
1¥
No
Filter cube
Selfaligning
Vert & horiz
slits,
5 sizes
Dichroic filter
cube
CCD or
EM-CCD
1
No/yes
CCD
Stepper,
±10 nm
NA
No
Dichroic,
4 positions
~1 k fixed,
50 mm
Dichroics/
filters
CCD
CCD
Yes/no
CCD
Piezo,
±100 nm
50 : 1
No
Dichroic,
6 positions
preset, rect.
array
adjustable
preset/
adjustable
5 slits,
10–100 mm
Dichroics/
filters
4 hi-QE PMTs
4
Yes/yes
10 bits
Piezo,
±100 nm
Dichroics/
Filters,
3 emisson,
3 barrier
Dichroics/
Filters,
3 emisson,
3 barrier
CCD or
EM-CCD
2-camera port
No/no*
CCD
CCD or
EM-CCD
2-camera port
No/no*
CCD
NA
1¥
NA
1 dichroic,
exchangeable
by user
Selfaligning
50 mm, 20 k on
disk, ~1 k
/FOV
NA
1¥
NA
Dichroic,
3 positions
Selfaligning
50 mm, 20 k on
disk, ~1 k
/FOV
0.7–40¥
0.84¥
Yes
Dichroic,
4 positions
4 x,y,(z),
diameter
adjustable
3, 200 steps,
0.1–13 Airy
Units 10–1 k
mm
3 dichroics,
6 positions, +
spectral
detector, 10 nm
/channel
3/4 filtered
PMTs;&/or
diff. Grating
w/32 mPMTs
array,7
8
Yes/yes
8–12-bit
Microscope
10 nm,
Piezo,
±5 nm
0.7–40¥
0.84¥
Yes
Dichroic,
2 positions
2 x,y,
diameter
adjustable
2 dichroics,
6 positions
2 filtered
PMTs,
trans PMT
4
No/yes
8–12-bit
0.5–2¥
1.18¥
Yes
Achrogate,
line-mirror
on clear
2
adjustable
1, 200 steps,
0.1–13 Airy
Units
10–1 k mm
17 slits, 0.5–10
Airy units
detector
dichroic,
12 positions,
8 position
512 ¥ 1 linear
CCD
2
No/no
8–12-bit
Microscope
10 nm,
Piezo,
±5 nm
Microscope
10 nm,
Piezo, ±5 nm
blank
barriers
4
These numbers assume that the tube mag is 1¥.
Yokogawa scanners are manufactured by Yokogawa Electric (Tokyo, Japan), but retailed by a number of companies including, Andor Technologies (Belfast, UK), Solamere
Technology (Salt Lake City, UT), PerkinElmer (Downer Grove, Il), Visitech (Sunderland, UK).
6
It is possible to use 4 lasers with a quad, dichroic beamsplitter.
7
Transmission PMT and 4-channel non-descanned PMT detector also available.
5
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Detection channels with stepless tunable bandpass and PMT
Beam splitter or mirror for auxiliary emission outlet (optional)
Emission filter and polarization filter (rotatable) (optional)
Excitation pinholes (excpt. IR)
Merge module. Combination of up to 4 visible lasers
a) Multiline Ar-Laser (457 – 476 – 488 - 496 – 514)
b) HeNe Laser 543
c) HeNe Laser 594
d) Kr Laser 568
e) HeNe Laser 633
f) IR Laser TiS for
Multiphoton excitation
g) HeCd Laser 442
h) Solid state Laser 430
i) Ar Laser 351 – 364
j) Diode Laser 405
EOM for intensity control of IR Laser
AOTF for intensity control on VIS and UV Lasers
Variable adaptation optics for UV / 405nm illumination
K-Scanning module for optically correct scanning method and field rotation
Scan lens
Beam splitter for non-descanned reflected light mode (optional)
Objective optics
Sample
Condensor optics
Detectors for non-descanned transmitted light (optional)
Secondary beam splitter for NDD transmitted light (optional)
Secondary beam splitter for NDD reflected light (optional)
Detectors for non-descanned reflected light (optional)
Beam splitter for UV illumination (optional)
Variable beam expander optics
Beam splitter for IR or violet illumination (optional)
Acousto Optical Beam Splitter (AOBS )
Pinhole optics
Detection pinhole
Spectral detector prism
FIGURE A2.3. Schematic diagram of Leica TCS SP2 AOBS. The Leica TCS SP2 AOBS is an
advanced confocal microscope in which all filtering and beam-splitting functions are performed
by either liquid-crystal or acousto-optical components. This makes the system extremely flexible
in terms of being able to add new lasers or adapt to new emission bands. The acousto-optical
beam-splitter (AOBS) is essentially transparent except at exactly the laser wavelengths (see Fig.
3.23). The K-scan galvanometer mirror arrangement is capable of being rotated around the optical
axis to change scan directions. There is one adjustable pinhole for all 4 prism/moving-mirror spectral-detection channels. Leica also makes the TCS SP5, which is similar but employes a tandem
scan system which permits one to switch between a scanner employing a normal, analog galvanometer and one employing resonant galvanometer for high-speed, bi-directional scanning at
up to 16 k lines/s.
1. TiS Laser (pulsed IR)
2. EOM for intensity control of IR Laser
3. K-Scanning Module for optically correct scanning
method and field rotation
4. Scan optics
5. Beam splitter for non-descanned reflected light mode
(optional)
6. Objective lens
7. Sample
8. Condensor optics
9. Detectors for non-descanned transmitted light
10. Secondary beam splitter for NDD transmitted light
11. Secondary beam splitter for NDD reflected light
(optional)
12. Detectors for non-descanned reflected light (optional)
13. Variable Beam expander optics
FIGURE A2.4. Schematic of the Leica MP RS Multiphoton Fluorescence Microscope. The Leica
MP RS is a single-beam scanning fluorescence microscope that uses a ps near-IR laser light source to
produce optical-section images of suitable specimens. It is designed for viewing living cells and incorporates a variety of non-descanned detectors to record both transmitted and backscattered fluorescence
signal. This instrument uses a fs-pulsed, near-IR laser multiphoton excitation and a high speed galvanometer to provide fast imaging. Figures kindly provided by Leica Inc. (Heidelberg, Germany).
Multi-anode PMT
SPECTRAL
DETECTOR
A
B
Unpolarized Light
P
Polarization
Rotator
Multiple Gratings
(2.5/5/10nm)
Polarized Beam
Splitter
SCAN HEAD
Optical
Fiber
SMA Connector
from Laser
Module
Galvanometer
Pair
Primary
Dichroic
Mirror
Mirror
Lens
3-COLOR
DETECTOR
Pinhole
Lens
Fixed
Mirror
Mirror
CH1
Emission
Filter
Emission
Dichroic 1
Nikon EF-4
Filter Block
PMT 1
Pinhole
Turret
CH3
Emission
Filter
Scan Lens
Emission
Dichroic 2
Nikon EF-4
Filter Block
PMT 3
Prism
CH2
Emission
Filter
Mounting
Adapter
PMT 2
to Microscope
FIGURE A2.5. Schematic of the Nikon C1si light path. The C1-Plus is a 3-channel fluorescence plus transmission, single-beam, galvanometer-scanned, confocal microscope. Because both the lasers and the PMTs are located externally and coupled through fibers, the C1 scan head is extremely compact and is very
easy to move from one microscope to another. The standard unit includes laser module, in which a wide variety of gas and solid-state lasers can be installed. a
scan-head and a DU-3 three-PMT detector module containing the collimating and focusing lenses, and photomultiplier tubes. The “si” version includes an additional sophisticated spectral detector that is also coupled to the scan head through a multi-mode fiber. The detector itself incorporates a Diffraction Efficiency
Enhancement System (DEES) in which a polarized beam splitter separates the unpolarized signal beam into two parts (red and blue lines). One part passes
through a prism polarization rotator so that all the light strikes the diffraction grating with the optimal (s-plane) polarization to be diffracted with maximum efficiency by one of 3 gratings (2.5, 5 and 10 nm/channel). Both ray bundles are then focused onto a 32 channel micro PMT by a pair of reflecting lenses (A and
B). Simultaneous readout is possible from all channels. The digitization system uses 2 sample-and-hold circuits to optimize signal integration. Figures kindly
provided by Nikon Inc. (Tokyo, Japan).
912
Appendix 2 • J.B. Pawley
PMT4
Emission beamsplitters
Emission fil t er s
Laser from optional
scanner
Confoc al
pinh ole
Grating
PMT3
Grating
Laser
port 1
Laser
port 2
Laser
port 3
PMT2
Slit
PMT1
Slit
S ca n n i n g
mir r ors
Confocal le ns
Pupil transfer lens
Excit ation
beamsplitters
To o bj ec tiv e le ns
Tube lens
A
FIGURE A2.6. (A) Schematic of the Olympus Fluoview 1000. The Fluoview 1000 is the most recent single-beam laser-scanning confocal fluorescence microscope introduced by Olympus. It offers 4 separate fluorescence detection channels, two of which incorporate diffraction gratings and adjustable slits to tune the
passband. Besides the normal scanning mirrors there is a second independent SIM-scanning arrangement (not shown in the figure) to control lasers used for
photo-uncaging or for intentionally bleaching the specimen. To keep the signal up when the light dose to the specimen must be kept low, this new scanner not
only incorporates dichroic elements employing “hard’ coatings to ensure the highest transmission, it also offers a photon-counting option to reduce PMT multiplicative noise. Figures kindly provided by Olympus Corp. (Tokyo, Japan).
Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2
913
Monitor
CCD camera
Imaging lens
Camera adapter
ND filter
Light
source
DSU
Fluorescent
mirror unit
Disk box
DSU
Rotary disk
Imaging lens
Illumination
tube
Light
illuminator
Objective lens
Specimen
B
W
L
C
FIGURE A2.6. (Continued) (B, C) Schematic of the Olympus DSU disk-scanner. The Olympus DSU is a disk-scanning confocal fluorescence microscope
that uses a mercury arc for excitation. The optical system is identical to that used for normal epi-fluorescence with the exception that an opaque disk is located
in the intermediate image plane. Slits in this coating on this disk allow light to reach the focus plane and prevents light from this plane from reaching the CCD
camera. To keep the light dose to the specimen low, this new scanner not only incorporates “hard” coatings to ensure the highest transmission of the dichroic
elements, it also offers a photon-counting option to reduce PMT multiplicative noise. (C) Layout of one of several interchangeable scanning disks used in the
Olympus DSU disk-scanner. The thickness and spacing of the slits varies on the 5 available disks have each been optimized for use with a particular objective.
Figures kindly provided by Olympus Corp. (Tokyo, Japan).
Micro lens array
Dichroic
Pinholes
Image
plane
Galvo scanner
A
FIGURE A2.7A. Schematic diagram of Visitech VT Infinity. The optical path starts with a stationary micro-lens array illuminated by an expanded laser beam.
A galvanometer mirror (x) incorporating a piezoelectric micro-deflector (y) scans the array to cover the sample and then de-scans the returning fluorescence
signal. This light is separated from the illuminating beam by a dichroic mirror, and passes through a stationary pinhole array to create confocal data. This data
is re-scanned, in perfect synchronization, by being reflected off the reverse side of the galvanometer mirror onto a sensitive CCD camera. The galvanometer
scanner is readily synchronized to the camera capture parameters, both exposure time and frame capture rate. Either multiple-line lasers or multiple lasers in any
combination can be coupled through an AOTF that provides high speed (~ms) laser-line selection and intensity control. Laser excitation can be coupled in either
by optical fiber or by direct coupling. Motor-driven filters change dichroic and detection bandpass. This system couples the advantages of high-brightness, laser
illumination with multipoint scanning to keep the instantaneous intensity down while providing a data rate high enough for fast image detection, using a highquantum-efficiency CCD camera. Figure kindly provided by Visitech Inc. (Sunderland, UK).
Primary
wheel
Fiber
Barrier
wheel
Negative
cylinder
Slit
Collimating
Positive
cylinder
AOD input
PMT
lens(e)
Scan
AOD
(astigmatism)
Field
B
FIGURE A2.7B. Schematic diagram of Visitech VT-eye. The VT-eye incorporates a novel acousto-optical deflector (AOD) scanner, that combines ultrafast horizontal scanning to provide high-resolution confocal imaging for
real-time, living-cell confocal microscopy. The AOD scans the X axis at up to
50,000 lines/s or 400 frames/s, fast enough to capture clear images of dynamic
events such as Ca++ puffs, sparks and waves. Multi-wavelength imaging for
multi-labeled specimens from UV through the visible to the near infrared is
achieved by using a selection of motorized, primary multi-band dichroics. The
system operates with almost any laser, or combination of lasers, and uses AOTF
technology to provide fast laser-line selection. The VTeye comes with up to 4
high-QE PMTs. The piezoelectric focusing system is capable of changing focus
positions at up to 100 slices per second. Although high-speed acquisition
creates vast quantities of data in a very short time, hours of experiments may
be recorded at the maximum capture rates on a range of parallel, hard-disk
modules. Figure kindly provided by Visitech Inc. (Sunderland, UK).
Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2
Laser for excitation
915
Camera port
Port select mirror
Barrier filter(triple)
Exciter filter(triple)
Eyepiece
ND filter & Shutter
Microlens array disk
Pinhole array disk
Microscope
Dichroic mirror
(triple)
excitation laser
Optical Path of CSU22
fluorescence
Microlens Disk
A
Pinhole Disk
Collimating
Lens
Fiber Input
Camera
Detector
Microscope
Port
Relay Lens
Relay Lens
Filter Wheel
B
FIGURE A2.8. Schematic of the Yokogawa CSU 22. The Yokogawa scanner was the first disk scanner to offer both laser illumination and multibeam excitation. The mircolenses increase the efficiency of the illumination path from the 2–10% common to ordinary disks to almost 60%. (A) Laser light enters the scan
head through a single-mode optical fiber, reflects off a mirror and through one of 3 exciter filters. After passing through an ND filter, and a beam expander, it
illuminates the microlens array on the top disk of the rotating scanning assembly. The lenses focus the light through a short-pass dichroic and onto the array of
pinholes in the lower disk. As this disk is in an image plane, the light passing each pinhole is focused into a point at the focus plane of the objective. Fluorescent light returning from the focus plane passes up through the pinholes, and reflects off one of 3 dichroic mirrors located between the two disks and into the
detection path. After passing through one of 3 barrier filters, a selection mirror sends this light either to the camera port or to the eyepiece. (B) Simplified ray
optical diagram of the CSU-22. The pinhole disk resides in an image plane and the signal passing the pinholes is first made parallel by a relay lens, then passed
through the emission filter before being focused onto the CCD chip by a second relay lens. Other details shown in Figure 10.9. Figures kindly provided by
PerkinElmer Corp. (Shelton, CT).
FIGURE A2.9A. Schematic diagram of Zeiss
LSM-5-LIVE Fast Slit Scanner. The LSM-5-Live is
a line-scanning confocal microscope using line illumination and a linear detector. Because it illuminates
about 100x more points than does a single-beam
instrument, the LSM-5 Live can acquire data at a
much higher speed while still keeping the peak light
intensity low enough to avoid singlet-state saturation.
In addition, the quantum efficiency of the linear CCD
is about 10x greater than that of most PMTs. Laser
light enters the scan head through optical fibers (1)
where it is combined by a series of mirrors (2, 3) and
then passes to beam shaper (an expander and a cylindrical lens that converts the collimated Gaussian
beam into laser light with a rectangular cross-section)
(4) and also focuses it precisely onto the AchroGate
beam splitter (5), reflects all wavelengths but only
along a reflective line across its center. As a result, no
matter what the wavelength, it reflects 100% of the
laser light but passes >95% of the signal light to the
detectors. The size of the raster on the specimen is
controlled by a 0.5–2x zoom optic (6), that feeds the
light to the y-scanning mirror (7), through the scan
lens (8), the objective lens (9) and on to the specimen, (10). Returning signal follows the same path but
mostly misses the reflective strip in the Achrogate and
proceeds through a wheel of secondary dichroic
beam-splitters (11) to one of 2 tube-lenses (12) that
each focuses the line illuminated in the specimen onto
a 17-position, slit aperture plate (13). Light passing
the slits is first filtered by emission filters (14) and
then detected by a 1 ¥ 512 linear CCD detector (15)
(see also Fig. 9.6).
A
M
Spectral Imaging
PMTA
G
PH
PMT
EF
PH
FO/
EPD
EF PH
laser
collimator lens
mirror
beam combiner
main dichroic beam splitter
SCXY
O
S
PH
scanner X/Y
objective
sample
variable pinhole
DBS
EF
PMT
G
PMTA
dichroic beam splitter
emission filter
photo multiplier tube
grating
PMT array (META)
NDD
FO
EPD
non-descanned detector
fiber out
external photodetector
DBS
PMT
DBS
Excitation
MDBS
L
CL BC
L
Imaging
L
CL
M
BC
MDBS
SCXY
M
NDD
O
S
B
FIGURE A2.9B. Optical beam path of the Zeiss LSM 510 META. A unique scanning module is the core of the LSM 510 META. It contains motorized
dichroic mirrors and barrier filters, adjustable collimators, individually adjustable and alignable pinholes for each of 3 (or even 4) detection channels, as well as
scanning mirrors, and highly sensitive PMT detectors including the 32 micro-PMTs of the META spectral detector. All these components are arranged to ensure
optimum specimen illumination and efficient collection of reflected or emitted light. The highly optimized optical diffraction grating in the META detector provides an innovative way of separating the fluorescence emission spectrum to strike 32 separate, micro-PMTs, each of which covers a bandwidth of ~10 nm.
Thus, a spectral signature is acquired at each pixel of the scanned image. Such a dataset can subsequently be digitally “unmixed” to separate signals from dyes
with overlapping emission spectra. The Beam Path: (1) Optical Fibers, (2) Motorized collimators, (3) Beam combiner, (4) Main dichroic beamsplitter, (5) Scanning mirrors, (6) Scanning lens, (7) Objective lens, (8) Specimen, (9) Secondary dichroic beamsplitter, (10) Confocal pinhole, (11) Emission filters, (12) Photomultiplier, (13) META detector, (14) Neutral density filter, (15) Monitor diode, (16) Fiber out.
Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2
T-PMT
HAL
917
VIS Fiber
Mirror
Collimator
Condensor
Specimen
UV Fiber
DBC
Objective
LSF NDF
Monitor Diode
VP3
EF3
PMT2
Inverted Microscope
Collimator
MDBS
DBS1
DBS3
VP2
EF2
VP1
EF1
PMT4
λ-selective
Element
Spectral
Detector
HeNe Laser
VP4
EF4
DBS2
PMT3
Tube Lens
Fiber
Coupler
AOTF
Shutter
HeNe Laser
Eyepiece
Fiber
Coupler
AOTF
Shutter
Ar/ArKr Laser
Tube Lens
Scan
Pinhole
Lens Scanner
Optica
x
y
MDBS
PMT1
HBO
Ar-UV Laser or
413 nm
Plate
Pinhole Optics
Scan Module on Side Port
TV
DBS1
EF1
VP1
Laser
Module UV
Laser
Module VIS
APD1
VP2
EF2
APD2
FCS on Base Port
APD Unit
C
FIGURE A2.9C. (Continued) Schematic diagram of Zeiss LSM FCS showing how the fluorescence-correlation spectroscopy (FCS) unit is attached via the
base port of the Axiovert 200M microscope while the LSM 510 META is attached to the side camera port. All figures kindly provided by Carl Zeiss Inc. (Jena,
Germany).
Appendix 3
More Than You Ever Really Wanted to Know About
Charge-Coupled Devices
James B. Pawley
INTRODUCTION
The electronic structure of crystalline Si is such that electromagnetic waves having the energy of light photons (1.75–3.0 electron
volts) can be absorbed to produce one free or “conduction” electron. If an image is focused onto a Si surface, the number of the
photoelectrons (PE) produced at each location over the surface is
proportional to the local light intensity. Clearly, all that is needed
to create an image sensor is a method for rapidly converting the
local PE concentration into an electronic signal. After almost 40
years of NASA and DOD funding, the slow-scan, scientific-grade,
charge-coupled device (CCD) camera is now an almost perfect
solution to this problem.
Success in modern biological light microscopy depends to an
ever-increasing extent on the performance of CCD cameras.
Because such cameras differ widely in their capabilities and are
also items that most biologists buy separately, rather than as part
of a system, some knowledge of their operation may be useful to
those practicing biologists who have not yet found it necessary to
be particularly interested in “electronics.” Although the basics of
CCD operation are described in many other chapters (particularly,
Chapters 4, 10 and 12) this Appendix describes the operating
principles of these devices in greater detail and also discusses the
ways that they “don’t work as planned.” It then covers the operation of the electron-multiplier CCD (EM-CCD), a new variant
that reduces the read noise almost to zero, although at the cost
of reduced effective quantum efficiency (QEeff).1 The second
section, How to choose a CCD, is a review of CCD specifications
with comments on the relevance of each in fluorescence
microscopy.
PART I: HOW CHARGE-COUPLED
DEVICES WORK
The first step is to imagine a rectangular area of the Si surface as
being divided into rows and columns, or more usually, lines and
pixels. Each pixel is between 4 ¥ 4 mm and about 24 ¥ 24 mm in
size and the location of any pixel of the surface can be defined in
terms of it being x pixels from the left side, on line y.
To construct an actual system like this, start with a smooth Si
surface; cover it with a thin, transparent, insulating layer of SiO2;
deposit onto the SiO2, a pattern of horizontal strips, made out of a
transparent conductor called amorphous silicon (or poly-silicon),
so that the strips cover the entire image sensor area. Although,
viewed from the top, these strips partially overlap each other, they
1
This loss can be avoided if the system is used in photon-conting mode.
are kept electrically separate from their neighbors by additional
layers of SiO2. Every third stripe is connected together to form
three sets of interdigitating strips that we will refer to as Phases 1,
2 and 3 (f1, f2, f3, Fig. A3.1). Taken together, all these phases constitute the vertical register (VR) and, after the assembly has been
exposed to a pattern of light, they are used to transfer the photoinduced charge pattern downwards, one line at a time. The pixels
along each line are separated from each other by vertical strips of
positively doped material injected into the Si. These positive
“channel blocks” create fields that prevent charge from diffusing
sideways without reducing the active area of the sensor.
Any photon that passes through the stripes and the SiO2, is
absorbed in the Si, producing a PE. If a small positive voltage (~15
volts) is applied to the f1 electrodes, any PE produced nearby will
be attracted to a location just below the nearest f1 strip (Fig. A3.2).
As additional PEs are produced, they form a small cloud of PEs
referred to as a charge packet. The number of PEs in the packet is
proportional to the local light intensity times the exposure period
and the problem now is to convey this packet to some location
where its size can be measured, and to do this without changing it
or losing track of the location from which it was collected. This
will be achieved by using the overlying electrodes to drag the
charge packet around in an orderly way until it is deposited at the
readout node of the charge amplifier.
Charge Coupling
The dragging mechanism operates in the following way: First f2
is also made positive so that the cloud diffuses to fill the area
underneath both f1 and f2. Then f1 is made zero, forcing the packet
to concentrate under f2 alone (Fig A3.2).
So far, these 3 steps have succeeded in moving the charge
packets that were originally under each of the f1 electrodes downwards by one phase or 1/3 of a “line” in the x-y raster. If this
sequence is now repeated, but between f2 and f3 and then again
between f3 and the f1 belonging to the next triplet of strips, packets
will have moved down by the one entire raster line. PEs created
within a particular pixel of each horizontal stripe remain confined
by the channel stops as they are transferred to the next line below.
A pixel of the image is therefore defined as the area under a
triplet of overlying, vertical charge-transfer electrodes and
between two neighboring channel blocks. The pixels on scientific
CCDs, are usually square, 4 to 30 mm on a side while those on
commercial, video CCDs are likely to be wider than they are high,
to conform with the reduced horizontal resolution of commercial
video standards. Only square pixels can be conveniently displayed
in a truly digital manner. Larger pixels have more leakage current
(dark-current), but are also able to store more charge per pixel (see
Blooming, below).
James B. Pawley • University of Wisconsin, Madison, Wisconsin 53706
918
Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006.
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
BASIC CCD ARRAY
Vertical
phase
One pixel
Control electrodes
Φ1
Φ2
Φ3
Drive pulse
connections
Image
section
Channel stop
Parallel channel
Serial channel
Readout
section
Output
Readout node
Φ4 Φ5 Φ6
Horizontal phase
Drive pulse connections
FIGURE A3.1. Layout of CCD array, viewed en face.
A
B
FIGURE A3.2. Charge coupling: Three stages in the
process of moving a charge packet initially beneath
phase 1 (A), so that it first spreads to be also under phase
2 (B) and finally is confined to entirely under phase 2
(C). These 3 steps must be repeated 3 times before the
charge packet has been moved downwards (or in the
diagram, to the right) by one line of the CCD array.
C
919
920
Appendix 3 • J.B. Pawley
At the bottom of the sensor, an entire line of charge packets is
simultaneously transferred to the adjacent pixels of the horizontal
register (HR, also sometimes called a shift register). Like the VR,
the HR is composed of a system of overlying poly-Si electrodes
and channel stops. Each column of pixels in the VR is eventually
transferred directly into the same specific pixel on the HR. The
three phases of the HR (f4, f5, f6) work exactly like those in the
VR, except that they must cycle at a much faster rate because the
entire HR must be emptied before the next line of packets is transferred down from the bottom line of the VR. In other words, in the
time between one complete line-transfer cycle of the VR and the
next, the horizontal register must cycle as many times as there are
pixels in each line.
At the right-hand end of the HR is a charge amplifier that measures the charge in each packet as it is transferred into it from the
last pixel of the HR. The first pixel to be read out is that on the
extreme right-hand side of the bottom line. The last pixel will be
that on the left side of the top line.2
The entire charge-transfer process has the effect of coding
position as time. If we digitize the signals from the charge amplifier, and store the resulting numbers in a video memory, we will
be able to see a representation of the light intensity pattern striking the sensor on any monitor attached to this video memory. Alternatively, as long as the dimensions of the CCD array match those
of some video standard, such as NTSC or PAL, the time sequence
of charge-packet readout voltages can be smoothed and, with the
addition of synch pulses, turned into an analog video signal. While
this latter process is often convenient, it is a poor plan if the analog
signal must then be re-digitized. The necessity to digitize twice can
reduce the effective horizontal resolution of the CCD sensor by
about a factor of 2 and because the process is AC coupled, photometric accuracy is severely compromised.
It is important to understand the relationship between the
charge-transfer electrodes and the charge packet. The electrodes
do not somehow “connect to” the charge packet, and “conduct” it
to the amplifier. Such a process would be subject to resistive losses,
charge would be lost and a lot of “wires” would be needed. The
charge-coupling process is better thought of in terms of a ball
bearing “dragged” over the surface of a loose blanket by moving
a cooking pot around underneath the blanket. The weight of the
ball and the lip of the pot create a dimple and gravity keeps the
ball in the dimple as the pot is moved. The voltage on the chargetransfer electrode creates an electronic “dimple.” Changing the
voltages on nearby electrodes moves the dimple. In this way,
groups of charged particles (electrons) can be pushed around
without actually “touching” or losing them.
Readout Methods
There are three distinct methods for reading out the charge pattern
of a CCD: full-frame, full-frame transfer and interline transfer
(Fig. A3.4). Most early scientific CCDs used the first method,
which operates as has just been described. Although full-frame
readout provides the largest sensitive area for a given area of
silicon, the lowest level of readout noise and the greatest photometric accuracy, it also has some disadvantages. One cannot both
collect and read out signal at the same time. Unless some sort of
shutter is used to prevent light from striking the sensor during vertical transfer, signal will be added to any packets that are trans-
ferred past bright features in the image, producing vertical streaking. This problem is more important when the exposure time is
short relative to the readout time.
In frame transfer readout, at the end of the exposure, the
entire charge pattern is rapidly (0.1–3 ms) transferred by chargecoupling to a second 2D storage array. The storage array is the
same size as the sensor array and is located next to it but it is physically masked with evaporated metal to shield it from light. The
charge pattern is then read out from the storage array while the
sensor array collects a new image. Because vertical transfer can be
much faster if the charge packets do not have to be read out, this
system reduces streaking by up to 1000¥ but does not eliminate it
and the need for a storage register reduces the fraction of the Si
surface area that can be used for sensing by 50%.
In interline transfer, the masked storage cells are interlaced
between the sensor cells (i.e., each pixel is divided into sense and
read areas). After exposure, all charge packets can be moved to the
readout array in less than a microsecond. This ability can be used
as an electronic shutter to eliminate vertical smearing but, because
at least half of the area of each sensor must be masked, and any
light striking a masked area is lost, the “fill factor” of the sensor is
reduced, proportionately decreasing QEeff. A solution to the “fillfactor” dilemma is to incorporate an array of microlenses, aligned
so that there is one above every pixel. With such a system, most of
the light striking any pixel will be focused onto the unmasked area.3
Although microlenses restore the QEeff somewhat, the full-well
signal possible is still limited by the smaller sensitive area.
WHAT COULD GO WRONG?
When I first heard the CCD story, it struck me as pretty preposterous! How could you get all the correct voltages (9 different
voltage combinations per pixel shift, ~3.6 million for each TV
frame, 108 million/s for video rate!) to the right charge-transfer
electrodes at the correct times? How could you get all of the charge
in a packet to stay together during a transfer? Wouldn’t Poisson
statistics apply, making even one transfer imprecise and the 2000
transfers needed to read out the top, right pixel of a 1000 ¥ 1000
pixel array impossibly inaccurate? How long would the PEs stay
free to be dragged around the lattice? Wouldn’t the charge packets
decay with time?
In fact, many of these problems did occur, but remedies to most
have now been devised. The difference between a $300 commercial CCD camera and a $65,000, top-of-the-line scientific CCD can
often be measured in terms of how many of these remedies have
been implemented. Therefore, it is worthwhile trying to understand
some of them so that one can buy what one needs. The following
discussion will define and discuss some of the more important
CCD technical specifications.
Quantum Efficiency
Quantum efficiency is the ratio of the number of impinging
photons to the number of PEs produced.4 Any photon with energy
in the range of 1–100 eV striking crystalline Si has a very high
probability of producing a PE. However, reflections and absorption by the overlying polysilicon electrodes,5 reduce the QE of
3
2
This may seem backwards until one remembers that any image of the real
world is usually focused onto the CCD by a single, converging lens, a process
that always inverts the image.
This occurs only as long as the initial angle of incidence is near to normal, a
condition met when CCDs are used for light microscopy.
4
In the visible range, each absorbed photon makes only one PE.
5
Kodak had pioneered the use of charge transfer electrodes made out of In and
Sn oxides that scatter less light than do those made of poly-Si.
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
921
FIGURE A3.3. Four CCD readout patterns: Full-frame,
frame-transfer, interline transfer and gain register (EMCCD).
front-illuminated CCDs especially in the blue end of the spectrum.
To reduce this effect, some UV-enhanced sensors are coated with
fluorescent plastics, which absorb in the blue and emit at longer
wavelengths. Others have their backs etched away and are turned
over to permit the illumination to reach the light-sensitive area
from the back side.6 Figure A3.4 shows the intrinsic QE of different types of CCD (not Qeff, which would take into account the light
lost if some of the sensor is covered by charge storage areas). The
effective QE can usually only be determined by actual measurement or by very careful evaluation of the published specifications
(QEeffective = QEintrinsic ¥ fill factor).
Edge Effects
In early CCDs, PEs were often “lost” in the crystalline imperfections that are always present at the Si/SiO2 junction. To avoid this,
ion implantation is now used to make an N-doped, sub-surface
layer called the buried channel about 1 mm below this surface (Fig.
A3.2). This channel attracts the free PEs, keeping them away from
the edge of the Si crystal. Any serious CCDs will have a buried
channel but the need for ion-implantation keeps CCD chip prices
high! Figure A3.5 shows the readout noise, in root-mean squared
(RMS) electrons/pixel, for surface and buried-channel CCDs
having two different pixel sizes. From this you can see that small
pixels (here ~5.5 ¥ 5.5 mm) have lower read noise than larger ones
(~17 ¥ 17 mm), mostly because the larger ones have higher capacitance and capacitance is the most important parameter of read-
6
Back-illuminated CCDs have to be thinned to 7–10 mm so that conduction
electrons created near what would have been the back surface can respond to
the fields created by the buried channel and the CC electrodes. Thinning
increases cost and also reduces QE at longer wavelength where the absorption distance of the photons becomes comparable with the actual thickness.
Back-illuminated CCDs are also more expensive because it is difficult to
create electrical contacts with electrodes, etc., that are now on the bottom side
of the chip.
amplifier noise. One can also see that at readout speeds higher than
1 MHz (or 1 second to read out a 1024 ¥ 1024 CCD), the read
noise increases with the square root of the read speed.
Charge Loss
The lifetime of a PE (before it drops back into the ground state)
depends on the purity and crystalline perfection of the Si and on
other factors such as temperature. Generally it is long enough that
little charge is lost during the exposure times commonly used in
fluorescence microscopy. If necessary, it can be increased by
cooling the detector, something often done to reduce dark charge.
Leakage or “Dark Charge”
Dark charge is the charge that leaks into a pixel during the exposure time in the absence of light. It can be thought of as the dark
current7 deposited into one pixel. Many processes other than
photon absorption can add PE to the charge packet. The magnitude of this dark charge depends on the length of the exposure, and
is substantially reduced by cooling. The rule of thumb is that for
every 8°C of cooling, the dark charge is halved. As noted above,
dark charge is principally a problem because it produces Poisson
noise equal to the square root of its magnitude, and if this is
left unchecked, it can significantly increase the noise floor of the
CCD.
Since ~1987, a process called multipinned phasing (MPP) has
been available to reduce dark charge build-up by about a factor of
1000, making it immeasurable in exposures up to a minute or so.
This feature should be specified if one expects to use exposures
longer than a few seconds without deep-cooling.
7
A current is a flow of charge measured in charge/time. The unit of charge is
the Coulomb (c). The unit of current is the Ampere (A). One Amp represents
a flow of one Coulomb/s or 6.16 ¥ 1018 electrons/s.
922
Appendix 3 • J.B. Pawley
FIGURE A3.4. Intrinsic QE as a function of wavelength for a front-illuminated CCD (blue), a visibleenhanced, back-illuminated CCD (green) and a
UV-enhanced CCD (red).
It should also be remembered that, while dark charge is never
good, its average value can be measured and subtracted on a pixelby-pixel basis, by subtracting a “dark image” from each recorded
image as part of flat-fielding. However, because, by definition
“dark” images contain very few photons/pixel, they have relatively
high Poisson noise and low S/N. Therefore, a number of such
images must be averaged to produce a correction mask that is statistically defined well enough that subtracting it from the data does
not substantially increase the noise present in the final, corrected
image.
This is not a problem when there are many counts in each pixel
because the subtractive process of dark-charge normalization
involves a change that is small compared with the intrinsic noise
present in a large signal. It can be a problem when the black mask
image is subtracted from a faint image that also contains only a
few counts/pixel.
What cannot be removed by flat-fielding is the Poisson noise
associated with the dark charge. This is equal to the square root of
the number of electrons/pixel it represents. CCDs should always
be operated such that the noise on the dark charge is less than the
readout noise. On conventional CCDs this condition can usually
be met quite easily by slightly cooling the sensor (0°C or about
-20°C from ambient). The use of lower temperatures is complicated by the risk of condensing atmospheric water, a process that
can be avoided only by enclosing the sensor in a vacuum chamber.
Generally, a vacuum-hermetic enclosure, combined with good
outgassing prevention, carries with it the significant benefits of
more effective cooling, long-term protection of the sensor from
moisture and other degrading organic condensates as well as
the prevention of front-window fogging. At video rate, where
exposures are short, dark charge is only a problem when the
readout noise is reduced to <1 e/pixel, as it is when an “electronmultiplier” (EM) charge amplifier is used (see below and also
Chapters 4 and 10). In EM-CCDs the read noise is so low that dark
current becomes the main source of noise and cooling to -80°C
becomes necessary.
Readout noise, electrons, rms
100
Measured
50
Surface channel
A = 300 µm2
30
20
Surface channel
A = 30 µm2
10
Buried channel (T<200K)
A = 300 µm2
5
3
Buried channel (T<200K)
A = 30 µm2
2
1
10 ns
100 ns
1 µs
100 MHz
10 MHz
1 MHz
10 µs
100 µs Clamp-to-sample time
100 kHz
10 kHz Pixel readout rate
FIGURE A3.5. CCD field effect transistor (FET)
noise as a function of pixel dwell time for large and
small pixels and when using buried channel vs. surface
channels. Smaller pixels have less read noise because
they have less capacitance. Buried channels have almost
10¥ less read noise than surface channels.
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
923
TABLE A3.1. Typical Performance of Various Types of CCD Cameras. The “Sensitivity” Column Is a Reasonable Estimate of
the Relative Suitability of the Camera for Detecting Very Faint Signals. It Spans a Very Large Range of Performance!
All CCDs are not equal
Type
Grade
QE % (effective)
Noise (e/pix)
Sensitivity (relative)
Bit depth
Dynamic Range
Video
commercial color
monochrome
1 Mhz, color
1 Mhz, mono
Back. Illum/
slow-scan
LLL-CCD
(EMCCD)
10
20
15
30
90
200
200
50
50
5
1
2
12
24
720
10
10
12
12
15
1,000
1,000
4,000
4,000
40,000
(18,000)*
?*
200,000
Digital
45
0.1
* Because the gain of the electron multiplier amplifier is unknown and large, it is not simple to measure, or even define, the sensitivity and bit depth of the EM-CCDs.
Blooming
As more photons are absorbed, the charge packet clustered around
the buried channel grows and mutual repulsion between these
electrons renders the field imposed by the charge-transfer electrode
ever less successful in keeping the packet together. The maximum
charge packet that can be stored without it overflowing into nearby
pixels can be estimated by multiplying the pixel area (in square
micrometers) by 600 PE/pixel (i.e., 27 k PEs for a 6.7 ¥ 6.7 mm
pixel, 540 k PEs for a 30 ¥ 30 mm pixel). This overflow problem is
referred to as “blooming” and, in CCDs for the home-video market
it is limited by the presence of an n-layer, deeper in the Si. When
the charge packet gets too big, mutual repulsion between the PEs
forces some of them into this overflow layer, through which they
are conducted to ground.
While this anti-blooming feature is convenient for removing
the effects of the specular reflections found in images from
everyday life,8 it is not incorporated into many full-frame or frametransfer scientific CCDs because it reduces QE for longwavelength light. As this light penetrates farther into the Si crystal
before being absorbed, much of it reaches the overflow layer where
any PEs produced are lost.
Incomplete Charge Transfer
Sometimes, an imperfection in the Si will produce a pixel that
“leaks” charge. Charge deposited into, or transferred through, this
pixel will be lost, producing a dark vertical line above it. In addition, if one pushes the pixel clock too fast, some PE in the packet
will not move fast enough and they will be left behind. In general
however, on a slow-scanned, scientific CCD, fewer than 5 PEs out
of a million are lost (or gained) in each, slower, vertical transfer
and only 50 (0.005%) are lost during each, faster horizontal transfer. In such devices, the main noise term is Poisson noise for any
signal level above ~20 PE/pixel,9 and it seems hard to imagine
doing much better than this except for signal levels <16 PE/pixel.
On the other hand, it is also true that the vast majority of CCDs
made (those for camcorders, surveillance cameras and even many
8
Features such as the image of the sun reflecting off a shiny automobile can
be over 1,000¥ brighter than the rest of the scene. Fortunately, such extremely
bright features are seldom found in microscopic images unless a crystal of
fluorescent dye occurs in the field of view.
9
This calculation assumes that the read noise is 4 e/pixel, and this will be less
than the Poisson Noise for any signal >16 PE. However, as many CCDs used
in microscopy have >4 e/pixel of noise, this cut-off point should not be considered inflexible.
scientific applications), operate with much (100¥?) less perfection.
In microscopy today, we find CCDs that span this range of performance (Table A3.1).
All CCDs are not equal!!
CHARGE AMPLIFIERS
So far, I have described an image sensor in which up to 90% of
the impinging photons make free PEs and explained how the
charge packets that result from many photons hitting a given pixel
can be conveyed to the charge amplifier, in a time-labeled manner
and almost without change. Clearly the performance of the entire
image detector will depend crucially on the capabilities of this
amplifier.
What Is a Charge Amplifier?
Although most scientists have had some exposure to electronic circuits that amplify an input voltage or current, they may be less
familiar with the operation of the type of charge amplifier found
in a CCD. The following outline is presented to enable the reader
to understand enough about the process to appreciate some of the
important differences between the various types of CCD.
Because of the pulsatile nature of the CCD charge delivery
system, the optimal way to measure charge packet size is to deposit
it into a (very) small capacitor (the “read node”) and then measure
the voltage on this capacitor with a high impedance amplifier. As
a field-effect transistor (FET) has an almost-infinite input impedance, it is ideal for this purpose and in fact, its existence makes
charge-amplification possible.
There are two basic types of conventional CCD readout
amplifier, non-destructive and destructive.10 Both employ FET
amplifiers.
Non-destructive (“skipper”) amplifiers use an FET with a
“floating gate” to sense the size of a charge packet by responding
to the moving field that is produced as the packet is transferred
along a charge-coupled register. Because the charge packet itself
is not affected by this process, the process can be repeated hundreds or even thousands of times. If the results of all these measurements are averaged, very low readout noise levels (>±1
electron/pixel) can be obtained, but at the cost of a substantial
10
The “electron-multiplier” amplifiers mentioned previously, act essentially as
pre-amps to the conventional FET amps described here. They will be covered
later in this Appendix.
924
Appendix 3 • J.B. Pawley
would, itself, produce a random electronic noise signal larger than
this, and electronic noise increases with readout speed, read-node
capacitance and, to a lesser extent, temperature.
The success of the CCD in overcoming this limitation depends
on two factors:
Reset
trigger
VReset
Supply voltage
Reset
transistor
Charge
input
∆V = Q
Readout transistor, Gain G
sampling
Cn
∆V = GQ
Output, G
Cn
Load
Q
CCD Register
Substrate and ground
Readout node
capacitance
FIGURE A3.6. Destructive read-out amplifier for a CCD chip.
(¥100 or ¥1,000) increase in readout time and logical complexity.
This approach might make sense on a Mars probe but it has not
been used in microscopy to my knowledge.
Destructive readout amplifiers are more common, probably
because they can operate more rapidly (Fig. A3.6). As implemented in a scientific CCD, the charge amplifier consists of the
following components:
• overlying charge transfer electrodes to drag the next charge
packet into the “read node”
• the read node itself: a 0.03–0.1 picofarad capacitor
• the sense FET
• the reset FET
In operation, fields from the overlying charge-coupling electrodes force a charge packet into the read-node capacitor, creating
a voltage, Vc, that is proportional to the amount of charge in the
packet. This voltage is sensed by the sense FET and the output is
passed, via additional amplifiers, to the analog-to-digital converter
(ADC) where the signal is converted into a digital number. Finally,
just before the next charge packet is coupled into the read node, a
reset FET discharges the capacitor, forcing Vc to zero, and allowing the read-FET to sense it again.
FET Amplifier Performance
The signal current (signal charge/s) coming from a CCD sensor is
very small. Suppose that there were, on average, 400 PE in every
pixel of a 512 ¥ 512 pixel sensor.11 Reading this out in one second
would constitute a current of only 10-11 Amps. The current through
the bulb in a home flashlight is 1010 times more. A very good conventional electronic amplifier designed to amplify this current
11
• The extremely small capacitance of the read node compared
to that of any other photosensor such as a photodiode.
• Special measurement techniques such as correlated double-
Although this number may seem small, it is actually quite high compared to
some uses in biological confocal microscopy. Many authors have found that
in normal” use, a single-beam confocal microscope used to image a fairly
faint stain will count 4–8 PE/pixel in bright areas of the image. Allowing that
the effective QR of a good CCD will be ~10¥ higher than that of the photomultiplier tube used in the confocal microscope, this makes the expected
peak CCD signal in an image from a disk-scanning confocal microscope only
40–80 PE/pixel.
Clearly there are a lot of tricks to making the perfect CCD
amplifier and not all CCDs employ them. Table A3.1 lists typical
performance for a variety of common camera types.
NOISE SOURCES IN THE
CHARGE-COUPLED DEVICE
Fixed Pattern Noise
When exposed to a uniform level of illumination, some pixels in
a CCD array will collect more charge than others because of small
differences in their geometry or their electrical properties. Consequently, it can be necessary to use stored measurements of the relative sensitivity of each pixel to normalize, or “flat field,” the final
dataset on a pixel-by-pixel basis. This is accomplished by first
recording an image of a featureless “white” field. This is often
approximated by a brightfield transmission image with no specimen, a process that will also record “inhomogeneity,” or mottle,
in the optical system. Differences in gain between pixels are
evident as visible as nonuniformities in the digital signal stored in
the memory and these are used to derive multiplicative correction
coefficients.12
Unfortunately, one can only preserve the high precision of the
CCD output if the coefficient used to normalize each pixel is
equally precise. In any event, these correction coefficients vary
with both the photon wavelength and the angle at which the light
passes through the polysilicon electrodes on its way to the buried
channel. This, in turn, depends on the details of the precise optical
path in operation when an image is recorded and may even change
with microscope focus! As the intrinsic noise of a pixel holding
360 k, PEs is only ±600 electrons or 0.16%, pixel-to-pixel normalization for changes in sensor gain is seldom perfectly effective
and consequently there is usually some level of “Fixed-pattern
noise” superimposed on the final data.
In addition, the “white” image that must be used for pixel-level
sensitivity normalization is itself subject to intrinsic noise (±600
electrons for a signal from a pixel with a full well charge of 360 k
electrons) and so multiplicative normalization may actually add
some noise to the raw, uncorrected signal! Fortunately, if the white
image can be defined by a multi-frame averages of several, nearly
full-well “white” images, this normalization noise should only be
noticeable when the image data to be corrected is similarly noise
free. Without details of the signal levels present or the optical
system in use, it is difficult to estimate the magnitude of normalization noise but it will be comparatively less important for images
of faint objects containing few counts/pixel because these measurements are themselves less precise.
12
These correction coefficients are small and only needed when operating on
images involving large numbers of photons (and consequently having relatively low Poisson noise and good S/N).
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
It should be also noted that the vignetting and “mottle” visible
in images characteristic of video-enhanced contrast microscopy
will produce small intensity errors in the data obtained by both
widefield and confocal. However, this noise term will be more
noticeable in widefield where more photons are used and hence the
precision of the data is greater. Mottle is produced by dirt and
surface imperfections on any optical components that are not
located exactly at aperture planes, as well as by non-uniformities
in the image sensor. Fortunately, to the extent that it is stable with
time, mottle will be removed by the flat-field correction for CCD
sensitivity just discussed.
What will not be removed is any change in signal caused by
stray light (room light, light that goes through filters designed to
remove it, etc.). The simplest test of any CCD set-up is to record
an image of “nothing” (i.e., room dark, no excitation, no specimen
etc.). Then do the same with 100¥ longer exposure time with the
room lights at your normal operating level. Now adjust the display
look-up tables so that you can “see the noise” in both the images
on the screen. Although the only difference between the two
images should be increased dark noise in the image with the longer
exposure, this is seldom the case.
Noise from the Charge Amplifier
Noise is generated by both the readout and the reset FETs in the
charge amplifier. Noise generated in the readout FET reaches the
ADC directly. If thermal noise in the reset FET prevents it from
completely discharging the read-node capacitance, it produces a
random offset at each pixel (i.e., the read-node voltage is not reset
exactly to zero). This is referred to as Reset Noise and has the
effect that the dark charge seems to vary from pixel to pixel. Fortunately, Reset noise can be almost eliminated by employing the
technique of Correlated Double-sampling (CDS) in the readout
amplifier. In CDS, the circuitry of the charge-to-voltage amplifier
is modified so that the output is proportional to the difference
between the value of Vc just after the reset pulse and its value after
the next charge packet has been inserted.
Although CDS essentially eliminates the effect of reset noise,
it also distorts the noise spectrum. On the one hand, this distortion
has the beneficial effect of converting the low frequency, 1/F noise
from the FET into broadband noise which is more easily treated
theoretically and which is less visually distracting than the short,
horizontal flashes characteristic of 1/F noise.13 On the other hand,
it means that the input to the ADC must be carefully frequencyfiltered. This filtering can be implemented either by employing RC
circuits or by using dual-slope integration (DSI) in the ADC itself.
If there are large intensity variations between neighboring pixels,
the use of RC circuits will effectively compromise the large
dynamic range of the CCD. Therefore, ADCs using DSI are
employed on most slow-scan scientific, cooled-CCDs.
The fact that CDS and, in particular, DSI work best at low
readout speeds is a final reason why most scientific CCDs operate
best at relatively low readout speeds (Fig. A3.5). The other two
reasons are improved charge transfer efficiency and the reduction
in broadband electronic noise from the FETs (noted above.)
13
In a CCD without CDS, noise features will seem to be smeared sideways,
while in one with CDS, they will appear as one-pixel-wide stipple with no
directionality.
925
Where Is Zero?
A final important feature of the CCD readout is that, compared to
the photomultiplier tube (PMT), it is relatively difficult to determine the exact output signal level that corresponds to a zero-light
signal. A properly operated PMT never records negative counts.
However, as the electronic readout noise of a cooled-CCD is an
RMS function with both positive and negative excursions, there
will be some pixels that measure lower than the mean value of the
zero-light pixel intensity distribution.
To ensure that no data is “lost,” scientific CCDs are usually set
up so that the zero-light signal is stored to be a few tens of digital
units (ADU) above zero. A histogram of numbers stored from a
“black” image will show a Gaussian-like peak centered at the
offset and with a half-width equal to 2¥ the RMS read noise (see
Fig. 4.20). This offset makes it more difficult to apply the gain and
offset normalization procedures to images that record only a few
detected photons in each pixel, a factor that will become more
important as CCDs are increasingly used to image living cells that
cannot tolerate intense illumination and which therefore produce
substantially lower signal levels.
A NEW IDEA: THE GAIN REGISTER AMPLIFIER!!
Early in 2002, a new type of readout amplifier was introduced by
Texas Instruments (Houston, TX) and E2V Technologies (Chelmsford, UK). As only E2V makes back-illuminated sensors, I will
describe their system but both work along similar lines. E2V originally referred to their device as the “gain register” and its purpose
is to amplify the size of the charge packet before it arrives at the
read node. Although the term gain register has recently been
replaced by the term “electron multiplier”, it is important to
remember that these new detectors work on a completely different
principle from that employed in intensified-CCDs.
The gain register superficially resembles an additional HR,
with two important differences:
• There
•
are 4 phases rather than the usual 3 and the new
phase consists of a grounded electrode located between f1
and f2.
The charge transfer voltage on f2, is now variable, between
+35 and +40 volts rather than the usual +15 volts.
As a result, when f2 is excited, there exists a high electric field
between it and the grounded electrode. The high field accelerates
the electrons in the charge packets more rapidly as they pass from
f1 to f2 with the result that each PE has a small (but finite; usually
in the range of 0.5% to 1.5%) chance of colliding with a lattice
electron and knocking it into the conduction band (Fig. A3.7).
Assuming the 1% gain figure, this means that for every 100 PE in
the packet, on average one of these will become two electrons
before it reaches the space under f2. Although this seems like a
trivial improvement, after it has been repeated as part of the 400
to 590 transfers in the gain register, a total average gain of hundreds or even thousands is possible. If the voltage on f2 is reduced
to normal levels, the sensor operates as a normal CCD.
As a result, a single PE can be amplified sufficiently to be
safely above the noise of the FET amplifier, even when it is operating at speeds considerably higher than video rate (35–50 MHz,
vs. 13 MHz for video). As the amount of gain depends exponentially on the exact voltage on f2, it is possible to “dial in” the
amount of gain needed to keep the signal level well above the noise
of the FET amplifier. However, it is important to remember that
926
Appendix 3 • J.B. Pawley
FIGURE A3.7. Energy diagram of an electron-multiplier CCD amplifier. The high field region that occurs between f2 and fDC when f2 goes strongly positive (right) causes about 1% of the electrons passing this region to collide with a lattice electron with sufficient energy to boost it to the conduction band. Repeated
over hundreds of transfers, this process is capable of providing an average amplification of hundreds or even thousands of times.
the use of high EM gain will tend to saturate the “full-well” capacity of later pixels in the gain register, reducing intra-scene dynamic
range.14 Although this effect can be reduced to some extent by
making each pixel in the gain register (and the read node) larger,
this approach is limited by the fact that one triplet of electrodes
can control a band of silicon only ~18 mm wide and because a
larger read-node capacitance increases the read noise of the FET
amplifier.
In sum, the gain-register CCD works like a normal fast-scan
CCD with no read noise. The high scan speed makes focusing and
sample scanning quick and easy and the device preserves the full
spatial resolution of the CCD because the charge packet from one
pixel is always handled as a discrete entity (unlike in an intensified-CCD). Of course, with fast readout, there is less time to accumulate much signal and the resulting image may have considerable
Poisson noise. But this is not the camera’s fault!
Alternatively, the output of many frames can be summed to
reduce Poisson noise or, if the signal is bright, one can turn off the
EM gain and have a fully functional scientific CCD.15
If the gain-register CCD is read out fast, there is so little time
for dark charge to accumulate that cooling would seem unnecessary until one remembers that one can now “see” even one PE of
dark-charge above the read noise. Because multi-pinned phasing
(MPP) is less effective during the readout clockings, significant
dark charge can be generated during readout. If the exposures are
short, this source of dark charge becomes significant, and in an
EM-CCD, even one electron is significant! In practice, the best
performance is obtained when the EM-CCD is cooled to between
-80° and -100°C.
14
If a register designed with enough pixel area to hold a normal full-well charge
of 30,000 electrons, is used with a gain of 100¥, then the pixels near the end
of the gain register will become full whenever the original charge packet has
>300 electrons.
15
Because, as noted above, the read node of the FET amplifier at the end of
the gain register in an EM-CCD has a relatively large capacitance, E2V offers
two separate FET readout amps. The one mounted at the end of the gain register is optimized for fast readout. The other is mounted at the end of the HR
not connected to the gain-register, has low input capacitance and is optimized
to read out slowly with low noise. Signal is sent to the latter by reversing the
charge transfer sequence applied to the HR.
EM-CCDs have one other important form of “dark noise”
called Clock Induced Charge (CIC, also known as spurious noise).
CIC typically consists of the single-electron events that are present
in any CCD, and are generated by the vertical clocking of charge
during the sensor readout. The process involved is actually the
same impact ionization that produces multiplication in the gain
register; however, levels are much lower because lower voltages
are involved. In conventional CCDs, CIC is rarely an issue as
single-electron events are lost in the read noise. However, in EMCCDs where the read noise is essentially zero and dark charge has
been eliminated through effective cooling, CIC is the remaining
source of single-electron, EM-amplified noise. If left unchecked,
it can be as high as 1 event in every 7 pixels. Fortunately, it can
be minimized by careful control of clocking voltages and by optimizing the readout process to cope with faster vertical clock speeds
(down to 0.4 ms/shift). This leaves a detector with less than one
noise pulse in every 250 pixels: a detector extremely well adapted
for measuring zero!
Of Course, There Is One Snag!
The charge amplification process is not quite noise free because
the exact amount by which each electron in the packet is amplified varies in a stochastic manner (i.e., some electrons are “more
equal” than others.). The statistical arguments are discussed in a
paper found at the URL listed below and in Chapter 4. In summary:
as the multiplicative noise inherent in the charge multiplication
process creates noise that has a form very similar to that produced
by Poisson statistics, the easiest way to think of its effect is to
assume that the amplifier has no noise at all but that the signal
being fed into it is half as big as it really is. In other words, the
camera will work perfectly but it will work as though it has a QE
that is only half of what it really is. Back-illuminated sensors are
now available with an intrinsic QE of about ~90% or ~45% when
used in the gain-register mode. This is 5–10¥ better than the performance available from most PMTs especially in the red end of
the spectrum.
It is worth noting that one can use electron multiplication and
still maintain the full QE by using the detector in photon-counting
mode, as is now being done by many astronomers. Photon counting is only possible when one is able to confidently see a single-
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
photon event as different from any dark event and when the
number of photons collected during an exposure is so low that
there is little probability of >2 photons falling into a pixel. To
implement photon-counting, one records a sequence of short exposures containing “binary”-type single-photon data, and combines
them to generate an image that is free of multiplication noise.16 To
be useful for recording dynamic events in living cells, an extremely
fast frame rate would be needed. This may be more possible with
some future EMCCD sensors.
(More info on EM-CCDs at http://www.emccd.com and
http://www.marconitech.com/ccds/lllccd/technology.html)
on back-illuminated chips, it can reach 90% (with somewhat
higher fixed-pattern noise).
The fill-factor is the fraction of the sensor surface actually sensitive to light. On the best frame-transfer CCDs, it can be almost
100%. On interline transfer CCDs it may be only 40%. Light not
absorbed in a sensitive area is lost, reducing the QEeff of the sensor
proportionally.
Factors affecting QE:
Front-illuminated chips
• Light
PART II: EVALUATING A
CHARGE-COUPLED DEVICE
•
•
A. Important Charge-Coupled Device Specs for
Live-Cell Stuff!
Although in Part I, much time was spent discussing cooled, slowscan, scientific CCDs, in fact, these have not been much used in
biological microscopy since Sony introduced the ICX085, 1 k ¥
1.3 k, micro-lens-coupled, interline-transfer chip in the late 1990s.
Although initially developed not for the scientific market but to
meet the needs of the Japanese high-definition TV standard, these
chips offered a set of practical advantages that biologists found
very appealing:
— As an interline transfer chip, it needed no mechanical shutter
and could be run so as to produce a continuous stream of
images.
— The high readout speed (up to 20 Mhz) allowed real-time imaging compared with the 5–10 s/frame readout then common.
— The 6.45 ¥ 6.45 mm pixels were small enough to sample the
image produced with high-NA 40¥ and 60¥ objectives.
— The 1 k ¥ 1.3 k raster size was both sufficient for most biological microscopy and significantly higher than that of the
other scientific chips then available.
— Mass production allowed the development of a micro-lens
array that increased the QEeff to an acceptable level for a frontilluminated, interline chip and did so at a price biologists
could afford.
As a result, the majority of CCDs sold for use in biological
microscopy today use this chip or its higher-QE cousin, the
ICX285. Although mass production made quality CCDs available
to many who formerly could not have obtained one, it is important to remember that the read noise of ±8–24 electrons/pixel
(depending on read speed) is substantially higher than ±2–3 electrons/pixel that characterized the best, slow-scan, scientific CCDs.
Although, as noted below, the difference is only important if the
dimmest pixel records fewer than ~50 electrons, and this seldom
occurs in widefield fluorescence microscopy, the disk-scanning
confocal fluorescence microscopes now available do provide an
image in which this difference is significant (Chapter 10).
•
•
• Sony has pioneered a process in which a micro-lens is mounted
above each pixel of a front-illuminated, interline-transfer
CCD. The lenses focus most of the impinging light onto a part
of the CCD where reflection losses are least, pushing the QE
to 65% in the green, less in the red (because of losses to the
overflow drain) and purple.
Back-illuminated chips
• Made by thinning the silicon and then turning it over so that
•
•
the light approaches the pixel from what would have been the
back side. This avoids scattering in the transfer electrodes and
increases the QE to about 90% in the green and >70% over the
visible range.
More expensive because of the extra fabrication.
Slightly less resolution and more fixed-pattern noise, caused
by imperfections in the thinning operation, and the presence
of two sets of surface states.
Color Chips
• One-chip color sensors employ a pattern of colored filters, one
•
QE is the ratio of photons striking the chip to electrons kicked into
the conduction band in the sensor. It should be at least 40% and
17
There is no multiplicative noise because any spike above the FET noise floor
counts as one electron, no matter how much it has been amplified.
is scattered by the transparent, polysilicon chargetransfer electrodes that overlie the photosensitive silicon
surface.
This scattering is more severe at shorter wavelengths. Light
that is scattered is not detected.
As blue light is absorbed nearer to the surface than red light,
and “deep electrons” may go to the wrong pixel, CCD resolution may be a bit lower than the pixel count at longer wavelengths, especially on chips with small pixels.
Best QE: ~20% blue, ~35% red/green
Two efforts to improve the QE of front-illuminated chips
include “Virtual Phase” (one phase “open,” Texas Instruments,
Houston, TX) and the use of indium oxides for the transfer
electrodes (Kodak, Rochester, NY). These have increased peak
QE to the range of 55%.
Micro-lens array chips
1. Quantum Efficiency (QE):
16
927
over each pixel. Light stopped by any such filter cannot be
detected and is therefore lost. The QE of such sensors is therefore at least 3¥ lower than for an otherwise comparable monochrome chip.
3-chip color sensors use dichroic mirrors to separate the
“white” light into three color bands, each of which is directed
to a separate monochrome CCD sensor. While this would seem
to ensure that “all photons were counted somewhere,” because
such systems seldom employ microlenses, their effective QE
is not much better than the 1-chip color sensors and alignment
of the signal light is important.17
While the QE is not much better, the resolution of the 3-chip camera is the
same as that of each chip, without the interpolation needed to disentangle the
3 colored images from the output of a 1-chip color sensor.
928
Appendix 3 • J.B. Pawley
• Color can be detected by making sequential exposures of a
TABLE A3.2. Dynamic Range and Pixel Size
monochrome chip through colored glass or LCD filters. This
produces the same QE losses as the patterned filter but has
the advantage that it can be removed when higher sensitivity
is needed. This design is not suited for imaging moving
objects.
Pixel Size
Full Well
Least significant bit =
Implied noise level
2. Readout Noise:
This spec is a measure of the size of the pixels and the quality of
the circuitry used for measuring the charge packet in each pixel.
It is measured in “±RMS electrons of noise” (i.e., 67% of a series
of “dark” readings will be ± this much).
14-bit camera
w/large pixels
6.7 ¥ 6.7 mm
27,000
6.5 electrons
±13 electrons
24 ¥ 24
345,000
21 electrons
±42 electrons
on the chip determines the total specimen-to-chip magnification needed!
• A good scientific CCD camera should have a noise level of
<±5 electrons at a readout speed of 1 M pixels/second.
• The readout noise increases with the square root of the readout
speed (see Table A3.2).
• NO Free Lunch! A chip that has ±5 e RMS of noise when
Two examples:
a. 1.4 NA 100¥ objective and a 1¥ phototube.
• The Abbe Criterion resolution @ 400 nm is about
0.22 mm. Magnified by a total magnification of 100¥,
this becomes 22 mm at the CCD.
• A CCD having 8 ¥ 8 mm pixels samples such an image
adequately (~2.8 pixels/resolution element).
b. 1.3 NA 40¥ objective and a 1¥ phototube.
• The Abbe Criterion resolution @ 400 nm is now
0.25 mm. Magnified 40¥ this becomes, 10 mm.
• A CCD having 8 ¥ 8 mm pixels is inadequate to sample
this lower-mag, high-resolution image.
readout at 100 k pixels/sec (or 10 seconds to read out a 1024
¥ 1024 chip), should produce ±50 e RMS of noise if read out
at 10 M pixels/sec (or 0.1 sec to read the same chip).
What Is “Good Enough”?
Very low readout noise is only essential when viewing very dim
specimens: luminescence, or low level fluorescence. Read noise is
only a limitation when it is more than the statistical noise on the
photon signal in the dimmest pixel (i.e., >sqrt of the number of
detected photons = sqrt # electrons).
Consider the signal levels that you plan to use. Will the darkest
important part of your image have zero signal or do you expect
some background signal from diffuse staining or out of focus light?
If the dimmest pixel in your image represents ~100 electrons, then
the Poisson or statistical noise on this background signal will be
±10 electrons. “Adding” an additional ±10 electrons of readout
noise will not make much difference to a measurement of this
background signal and it will be even less significant when added
to the even greater Poisson noise present in pixels where the
stained parts of the image are recorded.
This is especially true because RMS noise signals add as the
“sqrt of the sum of the squares” (i.e., the total noise from ±10 electrons of readout noise and ±10 electrons of Poisson noise is only
sqrt (100 + 100) = ±14 electrons).
On the other hand, if you are really trying to keep those cells
alive and you find that 2,000 electrons in the bright areas is enough,
the dark areas may now be only 50 electrons. As the sqrt of 50 is
about ±7, an additional ±10 electrons of readout noise may no
longer be acceptable, but only if you have to make measurements
in the dark areas on your image. In this case, the obvious choice
is a slower, quieter CCD or an EM-CCD.
While in widefield fluoresecence, the background stain level
is seldom so low that the sqrt of the signal recorded is lower than
the read noise, the disk-scanner does provide such an image
(Chapter 10). As one of the main advantages of disk-scanning is
that one can scan an entire image plane very rapidly, the fact that
one can read out the EM-CCD very rapidly without increasing the
read noise makes it the ideal detector for this type of scanner (or,
indeed for high-speed line scanning confocal microscopes).
12-bit camera
w/small pixels
If you must use this objective, you need either a higher mag
phototube (2.5¥) or a chip with 3 ¥ 3 mm pixels or (as CCD
pixels are seldom this small), some combination.
• Saturation signal level: The maximum amount of signal that
can be stored in a pixel is fixed by its area. The proportion is
600 electrons/square mm, so a 10 mm ¥ 10 mm pixel can store
a maximum of 60,000 electrons before they start to bleed into
neighboring pixels. In practice, as fluorescent micrographs of
living cells seldom produce signals this large, large pixels are
usually unnecessary.
However, the saturation level also represents the top end of
another spec, the dynamic range. This is usually quoted as
12-bit (4000 : 1) or 14-bit (16,000 : 1) etc., and represents the
ratio between the full-well saturation level and the readout
noise. Therefore, a camera with relatively high readout noise
can still look good in terms of dynamic range if it has large
pixels and hence a high full-well capacity. Conversely, a 12-bit
camera with small pixels can have less actual noise-per-pixel
intensity measurement than a 14-bit camera with large pixels.
In this case, the noise level of the 14-bit camera is >3¥ that
of the 12-bit camera. Your signal/pixel would have to be
3¥ larger in order to be “seen” when using this particular
14-camera.
4. Array Size:19 The argument for small
• Assuming 0.1 mm pixels (referred to the object plane), a 512
•
3. Pixel Size:
¥ 512 pixel chip will image an area of the specimen that is
about 51 ¥ 51 microns. If this is enough to cover the objects
you need to see, this small chip has a lot of advantages over
chips that are 1024 ¥ 1024, or larger.
Lower cost
• Nyquist sampling: The size of a pixel on the CCD is, in itself,
not very important BUT one must satisfy the Nyquist criterion: The pixels on the chip must be ~4+ smaller than the
smallest features focused onto it18 (see Chapter 4): Pixel size
18
19
Of two times smaller than the “resolution,” as defined by Rayleigh, or Abbe.
The array size refers to the number of lines and pixels in the sensor, not to
its total area.
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
• 4¥ fewer pixels to read out, meaning either:
•
— 4¥ slower readout clock, giving 2¥ lower readout noise.
— Same clock speed and noise level but 4¥ faster frame time.
(Easier to scan specimen to find the interesting part! Time
is money!)
4¥ less storage space needed to record data.
The argument for big:
• Manufacturing
•
improvements are reducing readout noise
levels at all readout speeds, and CCDs with more pixels often
also have smaller pixels which can lead to lower read noise. If
your labels are bright, having a larger chip allows you to see
more cells in one image (as long as they are confluent!).
Assuming that Nyquist is met in both cases, a large print of an
image recorded from a larger sensor always looks sharper than
one from a smaller array.
Binning: Binning refers to the process of summing the charge
from neighboring pixels before it is read out. This increases
the size of each charge packet read (making it look brighter)
and reduces the number of pixels. For example: 2 ¥ 2 binning
allows the owner of a 1024 ¥ 1024 chip to obtain the
speed/noise performance similar to the smaller chip (512 ¥
512) and to do so in a reversible manner. However, the optical
magnification may need to be increased to preserve Nyquist
sampling.
Before deciding that you need a larger chip, compare what you
would get if the same money were spent on another
scope/CCD/graduate student!
Bottom line:
• If more pixels means smaller pixels, they will each catch fewer
photons unless the magnification is reduced proportionally.
More pixels at the same frame20 rate mean somewhat higher
read noise because the pixel clock must go faster.
5. Readout Speed:
Although readout speed has been discussed above, we haven’t
mentioned that some good CCD cameras have variable speed readouts and the new EM-CCDs impose no high read speed penalty
(Table A3.3).
It is convenient to be able to read out the chip faster when
searching and focusing as long as one can then slow things down
to obtain a lower read noise in the image that is finally recorded.
However, the read speed is only one limitation on the frame rate:
TABLE A3.3. CCD Specifications
Array size
Pixel Clock Rate
Noise level*
Frame time
640 ¥ 480
13 MHz
(video rate)
100 kHz
1 MHz
5 MHz
100 kHz
1 MHz
5 MHz
200 e/pixel**
0.033
5 e/pixel
15 e/pixel
35 e/pixel
5 e/pixel
15 e/pixel
35 e/pixel
2.5 sec
0.25 sec
0.05 sec
10 sec
1 sec
0.2 sec
512 ¥ 512
1024 ¥ 1024
929
if the signal level is so low that 1 s/frame is required to accumulate enough signal to be worth reading out, then reducing the read
time much below 0.1 s loses some of its appeal.
Faster readout speeds are particularly important for moving
specimens, especially when doing widefield/deconvolution or
when following rapid intracellular processes, such as vesicle tracking or ion fluxes.
6. Shutter Stability:
Though not strictly a CCD spec, electronic (LCD) or mechanical
shutters are often built into modern CCD cameras.21 The latter have
the disadvantages of producing vibration and having a limited lifetime but the advantage that they transmit all of the light when they
are open (even an “open” LCD can absorb >50% of the light, other
electronic shutters may be better).
There seems little point in having a camera capable of recording (say) 40,000 electrons/pixel with an accuracy of ±200 e (or
0.5%!) if the shutter opening time is only accurate, or even reproducible, to ±10%. If one shutters the light source instead of the
camera, similar limitations apply.
7. User-friendliness:
State-of-the-art cameras often seem to have been designed to make
sure that no one unwilling to become a devotee of “CCD Operation” can possibly use them efficiently! Start off by asking to see
an image on the screen, updated and flat-fielded at the frame-scan
rate and showing as “white” on the display screen, a recorded
intensity that is only ~5% of the full-well signal. This is where you
should do most living-cell work. Then ask the salesman to help
you to save time-series of this image. Increase the display contrast until you see the noise level of the image, both before and
after “flat-fielding.” Put a cursor on one pixel in the top frame of
the stack and plot its intensity over the series.
8. “The Clincher” (Well, at least sometimes . . .):
Ask him/her what the intensity number stored in the computer for
some specific pixel means, in terms of the number of photons that
were recorded at that location, while the shutter was open. To
answer, the salesperson will have to know the QE, the fill factor
and the conversion factor between the number of electrons in a
pixel and the number stored in the computer memory (sometimes
called the gain-setting). To help them out, any “real” scientific
CCD camera has the latter number written, by hand, in the front
of the certification document (usually a number between 3 and 6).
If the salesman doesn’t understand the importance of this fundamental number, what hope is there for you? (Hint: It is important
because the Poisson noise is the sqrt of the number of electrons in
the well, not the sqrt of some arbitrarily proportional number
stored in your computer.)
Frame rate/s
30
0.4
4
20
0.1
1
5
B. Things That Are (Almost!) Irrelevant When
Choosing a Charge-Coupled Device for
Live-Cell Microscopy
1. Dynamic Range:
* Assumes conventional FET circuits. ** The readout noise is relatively higher at
video rate because the higher speed often precludes the use of various techniques,
such as correlated double sampling, that reduce readout noise.
This is the ratio of the “noise level” to the “full-well” (or
maximum) signal. Although 16-bit may sound a lot better than 12bit, you need to think before you are impressed.
The noise level should not be more than 5 electrons/measurement. Period!
20
21
The readout speed of a 2 ¥ 2 binned 1024 ¥ 1024 is a bit slower than an
actual 512 ¥ 512 because twice as many vertical clock cycles are needed,
and one still needs to read out pixel by pixel in the horizontal direction.
Often the same advantage can be gained by shuttering the light source. This
may become more common as pulsed laser or light-emitting-diode light
sources are introduced (see Chapters 5 and 6).
930
Appendix 3 • J.B. Pawley
Twelve bits is 4,000 levels. If the first level represents 5 electrons (in fact, it should represent half the noise or 2.5 e), then the
4,000th represents 20,000 electrons or (assuming a QE of 50%),
about 40,000 photons/pixel/measurement.
How often do you expect to be able to collect this much signal
from an area of a living cell only 100 ¥ 100 nm in size? You should
be able to get a good, 8-bit image using only 6% of the dynamic
range of a 12-bit CCD (Fig. A3.8).22
As the “full-well” signal is only proportional to the area of the
pixel on the chip (area in sq. mm ¥ 600), the dynamic range is only
really impressive if it is high AND a chip has small pixels. Then
it means that the readout noise is low. A test for actual dynamic
range is described below.
Bottom line: For disk-scanning confocal microscopy, a large
dynamic range is only important if it reflects a low readout noise
level.
Easier to just check the readout noise!
3. “Imaging Range” “Sensitivity” (or anything
measured in LUX):
2. High Maximum Signal (high, full-well
number, because of large pixels):
C. A Test You Can Do Yourself!!!
On living cells, you will probably never have enough light to reach
a full-well limit of even 20,000 electrons. Even if you do, there
are better ways to use it (more lower-dose images to show time
course?).
If the read noise is ±8 electrons, or 2
gray levels, one can obtain a
useful,“8-bit” (256 levels) image by
using only 6% of its dynamic range.
FIGURE A3.8. Not using the full dynamic range of a CCD. As most scientific CCDs have more dynamic range than one “needs” in live-cell fluorescence
microscopy, the excitation dose to the specimen can be reduced if one sets up
the CCD control program to display an 8-bit image using only the bottom 1,024
levels of a 12-bit image. Such an image is more than adequate for many functions in live-cell biological microscopy (particularly when other factors such
as dye-loading etc., may cause larger errors) and will require only 6% as much
signal as would a “full-well” image.
22
Remember, given optical and geometrical losses, you can collect no more
than about 3–10% of the photons produced, and, each fluoroscein molecule
will only produce perhaps 30,000 excitations before “dying.”
Stick to something you (and I?) understand: Photons/pixel or electrons/pixel. The other conversions are not straightforward.
4. “Neat Results”:
Unless you know how well stained the specimen is, you cannot
evaluate an image of it in a quantitative manner. (Though you
may not want to admit this!) By all means, view your own specimens, but viewing “test specimens” that are not expected to fade
and have a known structure (fluorescent beads in some stable
mounting medium?) facilitates A/B comparison. If you do use your
own test specimens to compare cameras, be sure to view them on
the same scope, and with the same conditions of pixel size and
readout time etc.
Better still . . .
Set up each camera that you want to evaluate on a tripod, add a
C-mount lens, and an ND 3 or ND 4 filter. Hook up a monitor or
computer and view some scene in your laboratory under ordinary
illumination (avoid light from windows which may vary from day
to day).
Close the lens aperture down until you can no longer discern
the image (see Fig. 4.20). This is the “noise-equivalent light level”:
the signal level at which the electron signal (i.e., photons/pixel ¥
QE) just equals the total noise level. Your measure is the aperture
at which the image disappears.23 Because it is sensitive to both QE
and readout noise level, this is a very useful measure of what we
all think of as the “sensitivity.” Of course, the signal level depends
not only on the light intensity but also on the exposure time and
the pixel area, so make sure to keep the former constant and make
allowances for the latter.
If you do not have even these meager facilities (a C-mount
lens, an ND filter, a tripod and some time), take an image of
nothing. Look at “no light” for one second, and for 100 seconds.
Ask to see a short line profile that plots intensity vs. position along
a line short enough that one can see the intensity of each individual pixel. The difference in the average intensity between the short
and long exposure is a measure of the leakage.24 With a little calibration from the published full-well specs (a spec less open to
“interpretation” than “noise”), you can even get a direct measure
of the read noise level from these dark images. (It should be the
standard deviation of the values as long as they are counted in electrons, not “magic computer units” and as long as fixed-pattern
noise is not a factor.) And just trying to work it all out will give
you some idea if the salesman knows anything . . .
D. Intensified Charge-Coupled Devices
Intensified CCDs (ICCDs) are just that: the mating of an “image
intensifier” to a CCD. The idea is that the photon gain of the intensifier (can be 200–2000¥) will increase the signal from even a
23
If the lens doesn’t have a calibrated aperture ring, you can open the aperture
all the way and reach the “threshold” exposure level by reducing the exposure time and adding ND filters. Remember to also correct for pixel area.
Larger pixels intercept more photons.
24
With a good EM-CCD, this measurement can be done using a short
exposure and high EM gain, then counting the number of amplified dark
charge/CIC spikes across a typical line of the raster.
More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3
single photoelectron above the read noise of the CCD. This occurs,
and can be particularly useful where fast readout is needed such
as when measuring ion transients. Finally, pulsing the voltage on
the intensifier section makes it possible to shutter (“gate”) the
camera on the ns time scale, making the ICCD useful for making
fluorescence lifetime measurements (Chapter 27, this volume).
However, ICCDs do not have the photometric accuracy of
normal CCDs for a number of reasons:
• The
•
•
•
25
relationship between number stored in memory and
the number of photons detected is generally unknown and
variable.
The intensifier photocathode has low QE25 (compared to that
of a back-illuminated CCD).
The “resolution” is generally only dimly related to CCD array
size because of blooming in the intensifier. To check this,
reduce light intensity until you can see the individual flashes
produced by single photoelectrons. See how many lines wide
they are. (They should be one line wide.)
They have additional noise sources: phosphor noise, ions in
intensifier section create flashes, high multiplicative noise in
the intensifier section greatly decreases QEeff, etc.
And the GaAsP photocathode with better QE, have to be cooled, making the
assembly very expensive.
931
• Photocathode resistivity can produce “dose-rate” effects: non-
linearities in which the recorded intensity of the brightest areas
may depend on (and affect) the brightness of nearby features.
Because I expect that EM-CCDs such as those mentioned
above will soon supplant ICCDs except where fast gating is
needed, I have not gone into more detail here. For more info, go
to: http://www.stanfordphotonics.com/
ACKNOWLEDGEMENTS
The author would like to thank Dr. J. Janesick, formerly of the Jet
Propulsion Lab (California Institute of Technology, Pasadena,
CA), for many conversations about CCD operation and for the
original sketches for Figures A3.1, A3.4, and A3.5 and to Colin
Coates, (Andor Technologies, Belfast, UK) for his helpful comments on the manuscript and for Figure A3.6.
REFERENCES
Inoue and Spring, 1997, Video Microscopy, Second Edition, Plenum, New
York, 1-741, particularly Chapters 5–9.
Pawley, J.B., 1994, The sources of noise in three-dimensional microscopical data
sets, Three Dimensional Confocal Microscopy: Volume Investigation of
Biological Specimens, (J. Stevens, ed.), Academic Press. New York, 47-94.
Index
I suppose it is inevitable that indexes are compromises: If one includes every mention of every entry, the index becomes as long as the
book. There is also the time dimension: As one cannot start writing the index until the book has been paginated, every day spent on
the index directly delays the publication date. For the Second Edition, I prepared the index somewhat in parallel with the page proofs
and it took most of a semester. For this Third Edition, a professional indexer was used to compile the initial index. We then expanded
the level of cross-referencing through a series of digital searches. The final result may show its mixed parentage.
As you use this index, please consider the following. I confess that many entries contain far fewer referents when they appear as
sub- or sub-sub-heads than when they appear as capitalized headings. In addition, some See alsomarkers use acronyms and it is also
true that these can get confused with the real title of the entry. In compensation, have tried to put in bold type those page numbers
on which I one would find the more comprehensive discussions of the topic we have added a period at the end of the major heads to
distinguish them from sub-heads. My apologies for any errors.
My thanks to Helen Noeldner for her calm and competent assistance during this long and laborious process. Please use the Feedback page at http://www.springer.com/387-25921-X to bring errors to our attention so that they can be corrected in future printings.
Remember that this Handbook has always been a community project. Good hunting!
JP, 2/21/06
Numbers
2D imaging, blind deconvolution approach,
476–477.
2D-time vs. 3D-time, embryo, 762–764.
2D pixel display space, 291.
2DCHO dataset, 818.
2DHeLa dataset, 818.
2-photon, (2PE). See Two-photon excitation.
3D Constructor, 282.
3D imaging, alternative approaches,
475–476, 607–624. See also,
Confocal topics; Multidimensional
microscopy topics.
episcopic fluorescence image capture
(EFIC), 607–608
light sheet microscopy (SPIM), 613
magnetic resonance microscopy (MRM),
618–624
amplitude modulation of RF carrier,
620
applications, 623–624
basic principles, 618–619
botanical imaging, 624
developmental biology, 624
Fourier transform, image formation,
620
future developments, 624
hardware configuration, 621, 622
histology, 623, 624
image contrast, 622–623
image formation, 619–621
Larmor frequency, 620
phenotyping, 623
schematic, 619
strengths/limitations, 622
micro-computerized tomography
(Micro-CT), 614–618
contrast/dose, 614–615
CT scanning systems, 615–618
dose vs. resolution, 616
layout, 614
living mouse, 615, 617
mouse femur, 616
operating principle, 614
tumor-bearing mouse image, 617
optical coherence tomography (OCT),
609–610
human retina 609
schematic, 610
Xenopus laevis embryo, 610
objectives on a tandem scanner, 154,
304
optical projection tomography (OPT),
610–613
lamprey larva, 612
mouse embryo, 612
plants, 774–775
setup, 611
real-time stereo imaging using LLCD
related methods, 607–625
selective plane illumination microscopy
(SPIM), 613
Medaka heart, 614
surface imaging microscopy (SIM),
607–608
3D Scanning Light Macrography, 672.
3D for LSM, 282.
3D methods compared, 448–451, 644–647.
table, 647
3D multi-channel time-lapse imaging
(4D/5D). See also, Time-lapse
imaging.
table, 384.
3D3T3 high-content screening dataset, 820,
821.
3DHeLa high-content screening dataset,
820, 821.
3PE. See Three-photon excitation.
4D imaging. See Four dimensional imaging.
4Pi microscopy, 561–570.
4Pi-PSF, 570
axial resolution, 563
I5M, 561, 569–570
OTF, 569–570
living mammalian cell imaging, 564–565
Golgi apparatus, image, 566
lobe-suppression techniques, 561
interference of excitation and detection,
561
confocal detection, 561
two-photon excitation (2PE), 561
MMM-4Pi microscopy, 554, 556,
563–564
basics, 565
scheme, 563
optical transfer function (OTF), 562, 563
outlook, 568–569
point spread function (PSF), 562–563
signal-to-noise ratio, 561
space invariance of PSF, 457, 490, 564
theoretical background, 562–563
type C, with Leica TCS, 4Pi, 565–568
imaging of living cells, 568
lateral scanning, 567
mitochondrial network, image, 568
optical transfer function (OTF), 567
resolution, 567
sketch, 566
thermal fluctuations minimized, 567
z-response, 563
5D image space, display, 291–294.
2D pixel display space, 291
animations, 292–293
color display space, 291
efficient use, 292
image/view display options overview,
table, 293
933
934
Index
5D image space, display (cont.)
multiple channel color display, 292
optimal use, 293–294
pseudo-color, 173–175, 190, 291
stereoscopic display, 293
true color, 291
A
Abbe, Ernst, 1, 5.
Abbe refractometer, 377.
Abbe resolution criterion, 36, 37, 60, 61,
65–68, 574, 575, 631–636, 928. See
also, Rayleigh criterion.
breaking the Abbe limit, 573
calculation, 65–66
individual point features separated by,
68
pixel size, 62, 65, 634–635, 784, 928
Abbe sine condition, 151, 239.
Abbreviations, list, 125.
Aberrations, 109, 146–156, 241, 411–412,
471, 480–481, 542, 629, 640–641,
654, 655, 657–659, 747. See also,
Chromatic aberrations; Refractive
index mismatch; Spherical
aberration.
astigmatism, 145, 151–152, 245–247,
249, 483,
axial, 242, 505, 542, 630
chromatic, 152–156, 160, 177–178, 209,
242–243, 641, 659
in 2-photon disk scanning, 542, 550,
554
of AODs, 56
axial chromatic registration, 287, 658
chromatic registration, 657–658
of collector lenses, 657–658
intentional, for height measurement,
224
magnification error, 155, 287, 331, 493,
542, 641, 657, 883, 904
measurement of, 243–244, 654, 659
multi-photon microscopy, 542
of optical fibers, 504, 507
signal loss, in confocal, 156, 178, 542,
641
standards, table, 157
coma, 145, 151–152, 245–246, 249, 483,
630
detecting, 241
monochromatic, 147–152, 542. See also,
Spherical aberration
optical, avoiding with thin disk lasers,
109
of refractive systems, 146–156
signal loss, 156, 178, 542, 641
spherical, 15, 34, 147–149, 151, 160, 192,
208, 241, 244, 247, 330, 395,
404–413, 454–455, 463, 466, 480,
542, 629, 640, 654–655, 657, 658,
728, 772, 774. See also, Spherical
aberration; Mismatch, refractive
index
blind deconvolution to remove,
480–481
cause of signal loss, 330, 389, 395,
413, 457, 542, 661
chapter, 404–413
correction for refractive index
mismatch, 192, 287, 411–412, 542
corrections for, 145, 411–412, 654–655
corrector optics, 192, 395, 398, 411,
477, 640, 655, 657
deconvolution, 463, 466, 468, 469, 471,
480, 498–499, 784
generated by specimen, 192, 418,
454–455, 654, 658, 747, 772, 775
of GRIN lens, 108
for IR wavelengths, 160
measurement using small pinholes,
145, 407
monochromatic, 147–151
multi-photon excitation, 542, 407–410
PSF, 148, 407, 455, 471, 481, 492, 657
secondary, 247, 249
in thick embryo imaging, 747
Zernicke coefficients, 247, 248
wave-front, measuring performance, 145
Ablation, 2-photon, 107, 764–765.
Absorber, saturable-crystal, 107, 111, 112.
to cover gap in titanium:sapphire lasers,
112
indium-gallium arsenide, InGaAs, 111
Absorption, 25, 163, 309–312, 338–339,
341, 514–518, 542, 550, 613, 704.
2-photon, 405, 535–536, 541, 545, 550,
552, 705, 719, 764, 884
caged compounds, 543, 544
CARS, 595–596, 599
contrast, 162–165, 211, 595, 610, 613,
770, 779
cross-section, 189, 426
energy levels, 514, 517, 682, 697, 705,
792
excited state, 544, 692
fiber optics, 501, 502
filters, 552
of fluorescent dyes, table, 345
fluorescent excitation, 45, 88
FRET, 184
and heating, 21, 218, 252, 539, 685
of incident light, 163, 177, 427
by ink, 73
and laser operation, 82, 108, 110, 116
light lost by, 25, 166, 414–418, 457, 654
lighting models, 283, 285, 309–312
molar extinction, 80–81, 343, 353, 357,
793
nonlinear, 188, 416, 427, 680, 704,
709–710
of optical materials, 158
and photodamage, 22, 685–686, 690, 750
in photodetectors, 253
photon, 550, 749
quantum dots, 221, 343, 357–358, 696,
759, 801
RESOLFT/STED, 573
self-absorption, 490
spectra, 217, 267, 338–339, 345, 355,
390, 415–416, 421, 538–539,
681–682, 706
in UV, 195
Absorption coefficient, complex specimen,
164.
Absorption contrast, 164–167, 195, 427.
equations, 164, 539
heating, 539, 685
Accuracy.
biological vs. statistical, 24, 36–37, 68,
312
position, 39–41
Acetoxymethyl ester indicators, 726.
deposits formaldehyde, 738
derivatization, 738
formula/reaction, 359, 738
loading method (AM ester), 358–359,
361, 726, 738–739, 744
painting brain slices with, 726–737
Achrogate beam-splitter/scan mirror, 50,
212, 231–232, 916.
operation, 50, 232, 916
Zeiss LSM5 line scanner, 212, 231–232,
916
Achromat, 152, 153, 244.
chromatic correction of, 153
flatness of field and astigmatism, 152
longitudinal chromatic correction, 153
measurement, 244
Acousto-optical beam splitters (AOBS), 45,
55–57, 88, 102, 211, 218, 395.
to select wavelength and intensity, 88,
102
to separate illumination and emission, 45,
218
Acousto-optical components, 43, 54–57.
tellurium oxide crystal, 55
thermal stability, 56, 57, 219
Acousto-optical deflectors (AOD), 25, 33,
54–56, 88, 447, 519, 543, 664, 762,
908.
as beam-splitters, to reduce loss, 33
to gate light source, 25. See also, AOM
group velocity dispersion due to, 88,
540, 646
multi-photon excitation, 88, 540, 543,
646
multi-tracking, 664performance, 55
problem descanning fluorescent light, 56,
447
Acousto-optical modulators (AOM), 11,
55–57, 88, 231, 519, 540, 543.
FRAP experiments, for controlling laser,
56
group velocity dispersion, 88
Acousto-optical tunable filters (AOTF), 43,
55–56, 88, 102, 219, 237, 346, 543,
651, 660, 673, 806, 908.
for selecting CW laser lines, 88, 102
blanking, 54, 55, 237, 389, 543, 628, 651
Index
leakage, 660
to regulate light intensity, 43
to spectrally filter light, 55
thermal sensitivity, 56–57, 219
Acridine Orange, 23, 344, 531, 665–667,
691, 774, 874.
bleaching, 693–694
Acronyms, list, 125.
Actin filament, 7, 236, 372, 378, 383, 692,
696, 714, 719, 748–749, 753, 756,
759–760, 773, 781, 804, 819,
824–825, 854, 856.
widefield source suitability, 142
Active laser medium, defined, 81.
Active mode-locked, pulsed laser, 111.
Actual focal position (AFP) defined, 405.
Actuator, galvanometer, 52.
Acute neocortical slice protocol, 723.
Adams, Ansel, zone system, 71–72.
Adaptive optics, 892.
ADC. See Analog-to-digital converter.
Adipocyte cells, CARS imaging, 604.
Adjacent fields, automated confocal
imaging, 810.
ADU, analog digital units, 74–77, 630, 925.
Advanced Visual System. See AVS.
Aequorea victoria, biofilms, 348, 356, 736,
794, 873–874, 877.
variants, table, 873, 874
Aequorin, Ca2+ reporter,736–737, 739, 741,
802.
developmental cellular application, 736
ion binding triggers light emission, 737
Ca++ signal detection, 737
AFP. See Actual focal position.
AIC. See Akaike Information Criterion.
Airy aperture, optimum for NA, 28.
Airy disks, 4, 24, 65, 131, 145–146, 151,
156, 210, 443, 444–449, 454–456,
463–465, 474, 485, 492–493, 562,
567, 630, 655–657.
Abbe criterion resolution, 65–66, 225
defined, 146, 444
diameter in image plane, 210, 225
four-lobed, from astigmatism, 151
image, 38, 146, 225
intensity ratios, 28, 145–146
inverse, 11
and line spacing, 24
radius and pixel size, 4, 24, 38, 39, 60,
65–67, 227, 485
vs. NA and wavelength, 1, 4, 146
Airy figure image, 38, 75, 79, 146, 147,
225, 479, 486–487, 562.
FWHM as optimal pinhole/slit size, 28,
36, 225, 232, 443, 454, 463–465,
564, 567–568, 630–631, 633,
655–657
and resolution, 65–67
size, and Nyquist criterion, 38, 39, 60
Airy unit, 28, 36, 41, 210, 222, 227, 232,
274, 443–451, 632, 775, 779.
Akaike Information Criterion (AIC), 825.
AlexaFluor dyes, 81, 103, 184–185, 190,
192, 236, 330, 342–344, 353–357,
360, 363, 393, 395, 416, 533, 540,
694, 726, 731, 749, 794, 799, 804,
810, 814, 854, 878, 880, 905.
fluorescence excitation, 355
living cells rapid assessment, table,
360
structure, 356
Alexandrite (Cr3+ in BeAl2O4), tunable laser,
109.
Alga.
autofluorescence, 357
autofluorescent image, 173, 175, 192,
194–195, 438–439, 528, 585, 785,
870, 881–885
biofilm, 870, 881–885
cell chamber for, 429
in laser cooling water, 116
Aliasing, 38–39, 271, 291, 293, 448, 588,
590–592, 640, 830, 833–834,
836–839, 903.
and Nyquist criterion, 38–39, 448
temporal, 39, 41, 391, 836–837, 839
Alignment, 25, 85, 134–135, 157, 505,
629–631, 651.
of laser systems, to reduce instability,
85
of optical coherence tomography, 610
of optical system, thermal stress, 85
importance, 25, 630
and PSF, 646
of source, 134–135, 629–631
Alkali vapor lasers, diode-pumped,
103–105.
Allium cepa. See also, Onion epithelium.
Alpha blending, 302, 304.
Alumina (Al2O3) ceramic tubes for lasers,
102.
Amira, 282–283, 286, 296, 302, 308, 312,
775–778.
Amoeba pseudopod, detail, 168.
Amplifier rods, maintenance, 116.
Analog digitization, for photon counting, 29,
33–37, 41, 65, 74, 78, 251, 254,
258–261, 263–264, 404, 460, 495,
522, 525–526, 542, 634, 766.
Analog-digital unit (ADU), to calibrate
CCDs, 74, 77, 630, 925.
Analog-to-digital converter (ADC), 31–34,
64–66, 70, 72, 74–75, 258–259, 261,
263, 286, 521, 630–632, 924–925.
Analyze (software), 281–282, 288, 290,
301–304, 312, 651.
Analyzer, in pol-microscopy, 25, 157, 229.
Analyzer, spectrum, 901–902.
Anemonea majano, sulcata, 874.
Angular deflection, distortion, 211.
Aniline Blue stain, 430–432, 435, 438, 774.
Animations, 281, 283–285, 289–290,
292–293, 295, 299, 308, 312, 764,
829, 835–839, 841–844.
Anisotropic crystals, 114.
935
Anisotropic sampling, 287–288.
when resampling, 833–835
Anisotropic specimens, 163, 286, 320, 329,
420, 623, 675, 678, 690, 710, 793.
Anisotropy analysis, chimeric proteins, 794.
Anisotropy of fluorescence, 742, 794.
Anisotropy of interference filters, 49.
Annular aperture, 4, 9, 20, 211, 889.
3D pattern of point-source from lens,
4–20
in specimen-scanning confocal
microscope, 9
Anti-bleaching agents, 36, 340, 363, 368,
375, 499, 694.
Antibody stains, 292, 339, 342–343, 348,
357–360, 375, 528, 576–578, 582,
610, 612, 664, 696, 731, 748, 760,
789, 802–804, 812, 852–855,
877–880.
artifacts, 664
biofilms, 877–880
FRET, 790–791
high-content screening, 812–815, 818
in situ, 612
penetration, 387
preparation, 369, 371–372, 375–377, 878
and TEM, 852–855
Antifade agent, 36, 340, 363, 368, 375, 499,
694. See also, Antioxidants.
Antiflex optics, to reduce reflections, 158,
171, 507, 513.
Antioxidants, living cell imaging, 341–342,
363, 389, 390, 729, 794.
Anti-reflection (AR) coatings, 1, 8–9, 25,
49, 117, 139, 145, 151, 158–159,
212, 505–506, 901.
color effect, 139
of optical fibers, 506
AOBS. See Acousto-optic beam-splitter.
AOTF. See Acousto-optic tuning filter.
APD. See Avalanche photodiode.
Apochromat, 15, 147–148, 151, 153–155,
158, 240–245, 409–410, 454–455,
655, 659, 771.
chromatic correction, 153
compared with fluorite objective, 154
longitudinal chromatic correction, 153
Apodization, high-NA objective lenses, 240,
243, 249–250, 272, 567, 889.
Applied Precision Instruments (API), 131,
137, 282, 388, 651.
APSS up-converting dye, saturation, 165.
AR. See Anti-reflection.
A. thaliana, 169, 173, 174, 175, 193, 196,
202, 416, 420–421, 423, 425, 426,
427, 431, 771, 772, 773, 775, 778,
779, 780.
attenuation spectra, 416
birefringent structures in cells, 420–421.
See also, Anisotropic specimens
bleaching, 203
double imaging, 169
fluorescence spectra, 421, 423, 425
936
Index
A. thaliana (cont.)
GFP protein fusion, 773
limitations for imaging, 772
mesophyll protoplasts, 196, 426
optical sectioning, 772, 775
protoplasts, 195–196, 203, 416, 421,
425–427, 429–430 438–439, 693
root tip fluorescence spectra, 173–175
seedling, autofluorescent image, 202
three-dimensional reconstruction, 190,
193, 771, 775, 777–778, 781
two-channel confocal images, 169, 175,
193, 196, 203, 427, 431, 772
two-photon excitation, advantages, 779,
780
two-photon fluorescence image, 427, 780
two-photon fluorescence spectra, 425, 426
Arc lamps, 132, 136–138.
current/stability of plasma, 138–139
monitoring during exposure, 137
radiance, 137–138
sensitivity to environmental variation, 136
shape of discharge, 132
shift of wavelength with temperature, 137
stability of, vs. filament lamps, 137
Area of interest. See also, Region of
interest.
identifying, 201–202
Argon-ion laser, 85–86, 90–102, 107,
109–110, 112, 119, 124, 203, 338,
341, 346, 353, 355, 375, 540–541,
655, 657.
CW, 90–103, 107, 109–110, 112, 119,
124
emission stability, 86, 102
references, 124
Argon-krypton mixed-gas laser, 90, 92, 93,
102, 108, 119, 203, 343, 375, 748,
798, 811.
Artificial contrast, vibration and ambient
light, 201–204.
Artificial lighting, image display, 306–312
Astigmatism, 145, 151–152, 245, 247, 249,
483, 505, 542, 630.
of AOD, 914
and flatness of field, 152
and intensity distribution, 152, 246, 630
laser optics, 89, 106–107, 505
measuring subresolution pinholes, 145
at off-axis points, 151, 245, 247, 249
ATP-binding cassette, 362.
ATP-buffer, 802–803, 812.
ATP-caged, 544.
ATP-gated cation channels, 359.
Attenuation of light.
by specimen, 164, 287, 298, 304, 320,
321, 414–418, 428, 439, 538, 558,
706, 779, 782
plots, 415, 706
of laser beams, 85, 87, 354, 415, 904
modeling, 309, 311, 320–321, 330
of PSF, 456, 462–463, 466, 494
x-ray, 614–615
Atto Bioscience CARV confocal
microscope, 215, 229, 230, 907.
Autofluorescence, 44, 81, 90, 173, 175, 195,
202, 339–340, 360–361, 369–370,
387, 414, 416, 421–434, 442–445,
447–449, 451, 509–510, 528, 530,
545, 607, 612, 663, 667–670, 678,
682, 690, 698, 706, 710–711, 713,
729, 742–743, 745, 764–765,
769–773, 779, 781–782, 785, 798,
815, 874, 876, 881–885.
of alga chloroplast, 168, 172–176, 202,
429–435, 556, 785
A. thaliana seedling, 202, 303, 307,
772
bleaching, 202, 698, 729
cell wall, 303, 431, 438, 770
emission spectra in plants, 176, 421–423
extracellular matrix, 311
fixation, as a cause, 358, 369–370
fluorescent probes, 339–340, 360–361
harmonic signals. See Harmonic signals
lamprey larvae, 612
multi-photon microscopy (MPM) See
also, harmonic signals, 545
optical materials, 45, 158
plants, 190, 193–195, 421–428, 770–772
plots, 176, 421–423
removal using spectral unmixing, 192,
382, 664–667
examples, 665–666
removal on basis of fluorescence lifetime,
345–346, 348, 349, 528
UV excitation, 347
Automated 3D image analysis methods,
316–335. See also, Automated
interpretation of subcellular
patterns.
biological objects, 319
blob segmentation example, 322–324
gradient-weighted distance transform,
323
model-based object merging, 323–325
watershed algorithm, 322–325, 777,
822
combined blob/tube segmentation,
328–330
data collection guidelines, 319–320
defined, 316, 328
future directions, 334
hypothesis testing, 318
illustrations, 317
image preprocessing, 320–321
background subtraction, 320
morphological filters, 320
signal attenuation-correction, 320–321
vs. manual, 316–317
montage synthesis, 282, 293, 312,
328–332, 748, 753, 851–852, 855,
858–859
defined, 329–330
examples, 330–332, 780–781
neuron, 330
scanning electron micrographs,
851–852, 855
TEM implementation, 858–859
neurobiology example, 320
quantitative morphometry, 331
rationale, 316
registration synthesis, 328–331
defined, 328
landmark-based, 328–329
multi-view deconvolution, 291, 330,
675–677
segmentation methods, 321–322
bottom-up, 321
hybrid, bottom-up/top-down, 322
integrated, 322
intensity threshold-based, 321
region-based, 321–322
top-down, 322
segmentation testing methods, 333–334
manual editing, 333–334
specimen preparation, 319–321
imaging artifacts, 320
stereology, 316
time series in vivo images, 319
tube-like object segmentation example,
324–328
mean/median template response, 328
skeletonization methods, 324–325
vectorization methods, 324, 326,
327
types, 318–319
Automated fluorescence imaging, 814.
endpoint translocation assays, 814
Automated interpretation of subcellular
patterns, 818–828. See also,
Automated 3D image analysis
methods 2D dataset analysis.
automated 2D analysis methods, 818
2D subcellular location features,
819–820
2DHeLa dataset images, 819
CHO cell dataset, 818, table, 820
Haralick features, 818–820
HeLa cells 2DHeLa dataset, 818
Zernike moments, 818–820
automated 3D analysis methods, 824
classification results, 824
feature normalization, 824
feature selection, 824
automated classification of location
patterns, 824–825
classification accuracy, 826
confusion matrix for 3DHeLa images
using SLF10, table, 824
confusion matrix for 3DHeLa images
using SLF17, table, 825
features in SLF17, table, 825
measured classification accuracy, table,
825
clustering of location patterns with
clustering consistency, table, 826
exclusion of outliers, 825
methods, 826
Index
optimal clustering determination,
825–826
optimal consensus tree, 827
clustering of location patterns, 825–826
downsampled images, different gray
scales, 824–825
future directions, 827–828
high-resolution 3D datasets, 820–822
3D3T3, 820
3DHeLa, 820
color images from 3DHeLa, 821
image acquisition requirements,
821–822
images from 3D3T3, 821
image database systems, 827
image processing/analysis, 822–823
3D SLF, 822–823
edge features, 823
feature calculation process, 822
morphological features, 823
segmentation of multi-cell images, 822
texture features, 823
protein subcellular location, 818
statistical comparison of patterns,
826–827
AutoMontage software, 282, 293, 304.
Avalanche photodiode (APD), 77, 233,
252–255, 404, 527, 542, 558, 567,
698.
array, for multi-beam sensing, 558
noise currents, 256
pulse pileup, 253, 527
unsuitability for non-descanned detection,
542
vacuum ADP, 254–255
Average intensity, 66, 110, 516, 556, 668,
684, 695, 747, 763–764, 816, 838,
930.
equation, 302, 309, 668
AVS (Advanced Visual System), 282–283,
286, 300, 311–311, 862, 863.
Axial chromatic aberration, 155, 658–659.
Axial chromatic registration, 154, 658.
Axial contrast. See z-contrast.
Axial edge response, 409–410, 654.
calculations for glycerol, table, 409
calculations for water, table, 409
Axial illumination, 60–61, 134.
Axial laser modes, 82, 110.
Axial minimum, 3D diffraction pattern, 4,
147.
Axial rays, spherical aberration, 148.
Axial resolution, 3–4, 6, 172, 182, 209, 211,
225–228, 230, 240–241, 243–244,
320, 370, 395, 407–411, 413,
444–446, 489, 493, 499, 511, 513,
551–553, 559, 561–568, 571–577,
610–611, 613, 649, 651, 654,
656–657, 659, 674, 704, 747,
750–751, 822.
4Pi microscopy, 561–568
coding, display, 305
defined, 3–4, 240, 444–446
focus shift, 243, 407–410
as function of pinhole diameter, 656
magnification, 215
measurement, 194, 656–657, 659
multi-photon, 750
multiview, 678
near focal plane, slit-/point-scan confocal
microscopes, 225–228
SHG, 704
SPIM, 614, 674, 751
STED, 571–577
tandem-scanning confocal microscope, 6,
225
tomography, 610–611
using mirror, 656–657
B
Back-focal plane (BFP), 34, 50–51, 58,
61–62, 84, 126–128, 166, 208–210,
225, 239, 268, 487, 509, 627, 629,
708.
Background light, from transmission
illuminator, 201–202.
Background noise, 260–262, 275.
Background signal, 12, 26, 28, 37, 68–69,
71–72, 88, 90, 112, 115, 158, 162,
168, 172–173, 175, 184, 188,
201–202, 221–225, 227, 232, 235,
248, 251, 257, 266–275, 278–279,
283, 287,-288, 290, 301–302, 305,
312, 321, 326, 339–340, 343, 345,
348, 360–362, 375, 421, 423,
428–429, 432–433, 442–451, 462,
465, 472–477, 486, 493, 497, 506,
509–510, 518–519, 535, 541, 543,
553, 559, 582, 584–585, 595,
598–600, 602, 604, 621, 633, 656,
663–370, 676, 694, 697, 698, 707,
713, 727, 733–734, 736, 747,
755–757, 760, 798, 801, 803, 809,
813, 815, 818, 822, 830, 836, 839,
851.
Background subtraction, 284, 301, 320, 473,
510.
Back-illuminated CCD, 31, 77, 222, 232,
234, 754.
Back-propagation neural network (BPNN),
818.
Backscattered light (BSL), 22–23, 57,
83–84, 130, 141, 145, 165, 169–170,
180–182, 191, 196, 202, 212, 221,
228, 240, 376, 378, 416, 430, 436,
442, 631, 879.
access to, antiflex optics, 6, 57, 141, 212,
229, 507, 513, 609, 631, 704, 707,
854, 879, 990
biofilm, image, 880
contrast, effect of specimen absorption,
165
effect of coherence on, 130–131, 170
images made using, 22–23, 154, 436–438,
513, 638, 855, 880
Amoeba pseudopod, 168–170, 191
937
cheek cells, 22, 23
diatom, 145, 438, 638–640, 881
latex bead, 182, 196, 197, 653
transparent ciliate protozoa, 141
LLLCD objectives/3D color-coded BSL
as a noise signal, 663
optical coherence tomography, 609
practical confocal microscopy, 631
from specimen, 202
unmixing, 192, 382, 664–667
Back-thinned CCD, 31, 77, 222, 232, 234,
754.
QE plot, 29
Bacteria. See Biofilms.
Ballistic microprojectile delivery, 360, 726,
803.
Ballistic photons, 418, 427, 538.
Ballistic scans, 40, 41.
Balloon model segmentation methods, 776.
Bandpass, optical filters, 43–44, 46, 48, 49,
51, 76, 87, 132, 141, 173, 204, 341,
528, 708, 798.
for CARS, 598–599
coupling short and long-pass filters, 46
excitation and emission, 48, 141, 217,
341, 708, 757, 798
laser, 106–107
liquid crystal, 425
to select range of wavelengths, 43–44
spectral detector, 203–204, 662–663,
666–667
Bandwidth, 32, 64, 69.
3 dB point, definition, 59, 65
of AOBS, 57
electronic/optical, digitization, 32, 34, 70,
238
head amplifier, 251
limiting, to improve reconstruction, 69
Nyquist reconstruction, output, 64, 69, 70,
238
Bead, fluorescence emission, 181, 182, 196.
fluorescent, 454, 477, 493, 499, 527, 652,
653, 656, 659, 784, 900, 904, 930
image, 656
table, 653
glass, in water, 181, 198–199
latex, fluorescence image, 196, 407,
455–457, 463, 471, 656
in water, confocal serial sections, 182
Beam blanking, 54, 55, 237, 389, 543, 628,
651.
Beam collimation, 728.
for fiber delivery, 506
Beam delivery, with fiber optic coupling,
85–88, 107, 216, 503, 506–508.
Beam deviation, unintentional, 15–16.
Beam expander, 8, 84, 124, 208, 212–214,
231, 650, 682, 708, 728, 907.
advantages, 213
Beam pointing, lasers, 85, 103, 107, 201,
250.
active cavity stabilization, 87
Beam quality, of diode lasers, 107.
938
Index
Beam shift, vignetting due to, 211.
Beam-splitter, 33, 46–48, 50–51. See
Dichroic mirrors.
Achrogate, 50, 212, 231–232, 916
AOBS, 56–57
broadband, 346
dichroic, 25, 33, 35, 43–51, 56–57,
83–84, 88, 139, 132, 135, 143, 151,
162, 203–204, 207–208, 211–214,
217–218, 229, 231–232, 266, 339,
341, 346, 375, 386, 424, 469,
503–504, 552, 563–564, 599,
630–632, 647, 650, 657–658, 664,
667, 691, 707–708, 747, 771–772,
810, 846, 879, 910, 907
table, 799
fiber-optic, 503–504
forty-five degree, performance, 47
fused-biconic coupler, 503–504
long-pass cut-off, 43, 46, 51, 175, 204,
564, 801, 875
multi-photon, 540–541
polarizing, 13, 50, 57, 85, 87, 100, 217,
513, 631
spectral problems, 50–51
triple dichroic, 33, 46, 48, 217–218, 658,
783
losses due to, 33
performance, 46–48
Beam scanning, along optical axis, 215,
555.
Beam-scanning confocal microscope. See
Confocal entries; Flying spot
ultraviolet (UV) microscope.
chromatic correction, 177
Beam-scanning systems, 6, 7, 16, 132, 146,
151, 156, 166, 177, 214–215, 218,
381, 554, 562, 564, 567, 568, 599.
coma in, 151
off-axis aberrations affecting, 156
Before-bleach/after-bleach ratio, FRET, 794.
Benchtop fiber-optic scanning confocal
microscopes, 507–508.
Bertrand lens, 61, 157, 412, 643.
Beryllium oxide (BeO), for laser tubes, 102.
Beta barium borate (BBO), non-linear
crystal for frequency doubling, 100,
109, 114–115, 125.
BFP. See Back-focal plane.
Bibliography, annotated, 889–899.
adaptive optics, 892
books on 3D light microscopy, 889
differential phase contrast, 892
display methods, 892–883
fiber-optic confocal microscopes, 883
general interests, 891
historical interests, 889–890
index mismatch, 893–894
multiplex, 894
non-linear, 894
point spread function, 895–896
polarization, 894–895
profilometry, 895
pupil engineering, 896
review articles, 889
technical interests, 891–892
theory, 890–891
thickness, 896
turbidity, 896–897
variants on main theme, 897–899
Binding equation, for fluorescent indicators,
740.
Biocytin, 730, 731.
EM imaging of brain cells labeled, 731
protocol, 730
Biofilms, 287, 688, 529, 530, 624, 779,
870–887.
2-photon imaging, 530, 882–885
dual-channel imaging, 884
limitations of CLSM and 2-photon, 884
single-photon/2 photon comparison,
883
thick environmental biofilms image,
885
autofluorescence, 545
backscattered light, 880
fluorescent proteins for, table, 874
future directions, 887
GFP variants for, table, 873
imaging extracellular polymeric
substances (EPS), 879–882
lectin-binding analysis, figures, 881,
882
lifetime imaging, 530
magnetic resonance microscopy, 624
making bacteria fluorescent, 873–874
pH imaging, 530, 739–745
sample mounting, 870–873
flow chamber system setup, 872–873
perfusion chambers, 870–872
pump selection, 871
upright vs. inverted microscopes, 870,
872
water-immersible lenses 149. 161, 209,
411, 429, 568, 613, 727, 737, 870,
872.
stains for, 874–879, 875
Acridine Orange, 23, 344, 531,
665–667, 691, 774, 874
antibodies, 877–878
biofilm community on tooth, 879
DAPI, 874. See also, DAPI
effect of antibiotic treatment, 877
embedding for FISH, 876–877
FISH with fluorescent protein,
875–876, 878
imaging bacteria, backscattered light,
879
live/dead stain, Streptococcus gordonii,
876
nucleic acid, 874–875
preparing labeled primary antibodies,
878
SYTO, 874–875
temporal experiments, 885–886
multi-cellular biofilm structures, 886
time-lapse confocal imaging, 885–886
transmitted laser light image, 880
Bioimagers, kinetics, endpoint analysis,
816–817.
Biolistic transfection, 360, 724–726, 803.
Biological accuracy, vs. statistical accuracy,
24, 36–37, 68, 73, 312.
Biological reliability, of measurements, 24,
36–37, 68, 73, 312.
Biological specimens, 6, 11, 12–13. See
also, Plant cell imaging, Biofilms,
Specimen preparation, and entries
under specific equipment and
cell/tissue type.
backscattered light images, 22–23, 25,
167–168, 170, 880
CARS imaging, 603–604
adipocyte cells, 604
epithelial cells, 603
erythrocyte ghosts, 603
distortions caused refractive index
inhomogeneity, 40–41, 181, 182,
198–199, 419
tandem scanning systems for, 6, 11
Yokogawa CU-10, 12–13
Biophotonic crystals, 188, 428.
Bio-Rad, 25, 33, 35–36, 70, 113, 214, 260,
630, 638–640, 657, 748–752, 757,
759–762, 858, 889.
1024ES, 710–711, 714, 718–719
data storage, 585
using white light source, 113
MRC 1024, photon counting, 33
photon efficiency, 25, 32, 261,
748–752
MRC-600 scanner, full-integration
digitizer, 70
PMT, 260–261
Radiance-2100, 23, 185
resolution, 657
Biosensors, fluorescent, 33–8348, 799, 805.
See also, Dyes, Fluorophores, and
Chapters 16 and 17.
future, 805
mitotic clock measurements, 799
Birefringence, 6, 15, 54, 83, 103, 109, 113,
116, 162–164, 188, 189, 414,
420–421, 431, 434, 436, 438, 479,
503, 710–711, 714, 717, 894.
acousto-optics, 54, 55
collagen fibers, 164, 188, 717
contrast, 15, 162–164, 188, 414–428,
431–438, 710–711, 714, 717, 719,
894
deconvolution, 479–480
defined, 163, 188
in fiber-optics, 503
harmonic generation from, 428, 431–438
images of Cymbopetalum baillonii, 189
in laser components, 85, 103, 109, 113,
116
quarter-waveplate, 6
table, 715
Index
Birefringent crystals, 188, 420–421.
optical effects of acoustic fields on, 54,
55
Black-body radiation, 44, 135–136.
from incandescent lamps, 44, 126,
135–136
spectrum, 136
Bleaching, 10, 12–13, 20, 24, 44, 63–64, 90,
142, 186–187, 194, 202–203, 210,
218, 220, 222, 340, 382–387, 442,
539–540, 690–702, 797, 905, 907.
2-photon excitation, 539–540, 680–689,
905
acceleration, 341
of acceptor in FRET, 184–187
anti-bleaching agents, 36. See also, Antibleaching agents
bleach patterns, 3D, 538, 628, 693
beam blanking, to reduce, 53–54
before/after ratio, for donor/acceptor pair,
794
chapter, 690–702
combining fluorescence with other,
383–386
in dye lasers, 103
dynamics, 202–203
fluorescence correlation spectroscopy,
383, 801
fluorescence lifetime, 382–383
fluorescence recovery after
photobleaching, 51, 54, 56, 80, 90,
187, 210, 218, 224, 229, 237, 362,
382, 684, 390, 691, 759, 801, 805,
850
FRET, 186, 382, 794–798, 800
fluorescence speckle microscopy, 383
in four-dimensional imaging, 222
improvement, recent, 36
laser trapping, 383
linear unmixing, 192, 382, 664–667
of living cells, 212, 220, 382, 797. See
also, FRAP, FLIP
intensity dependence, 341, 363
mechanism, 222–223
of non-specific fluorescence, 27, 44, 74
optical tweezers, 383, 385
performance limitations, 221, 224, 232,
381, 448–450, 556, 693. See Chapter
39
photoactivation, 187, 224, 383, 385, 541,
544–545, 693, 759
photo-uncaging, 383. See also, Photouncaging and signal per pixel,
63–64
spectral unmixing, 192, 382, 664–667
table, 384–385
techniques, 125
temperature as a variable, 696–698
time-lapse fluorescence, 382
Bleedthrough fluorescence, 185, 203, 664,
904.
multi-tracking, reduces bleed-through,
664
Blind deconvolution, 190, 468–487. See
also, Deconvolution.
2D approach, 476–477
3D approach, 475–476
advantages/limitations, 468–472
algorithms, 472–474
of A. thaliana seedling image, 190
confocal stack, 470
data collection model, 472
data corrections, 477
defined, 469
DIC schematic, 475
DIC stack example, 470
different approaches, 475–477
deblurring algorithm, 476
Gold’s ratio method, 476
inverse filter algorithm, 476
iterative constrained algorithms,
475–476
Jansson-van Cittert algorithm, 476
nearest-neighbor algorithm, 476
no-neighbor algorithm, 476–477
processing times/memory table, 476
Richardson-Lucy, 497, 568
TIRF microscopy, 477
differential interference contrast (DIC),
473–475
examples, 469, 470, 481, 482, 483
flowcharts, 473, 474
future directions, 483
Gerchberg-Saxton approach, 472
hourglass widefield PSF, 474
light source/optics alignment, 478
maximum likelihood estimation (MLE),
472–477, 669–670
new developments, 478–480
live imaging, 480
polarized light microscopy, 479
subpixel imaging, 478–479
optical sectioning schematic, 469
OTF frequency band, 474
simulated example, 481, 482
speed, 482–483
spherical aberration correction, 480–481,
471
spinning-disk confocal example, 481,
482, 482
transmitted light, bright-field (TLB), 472,
477
two photon example, 481, 483
widefield simulated example, 481, 469
WWF stack example, 469
Blind spots, due to sampling with large
pixels, 38.
Blue Sky Research, ChromaLase 488, 107.
Boar sperm cells, 557.
BODIPY dye, 142, 342–343, 353–356, 389,
692, 749, 760–762.
BODIPY TR, methyl ester dyes, 760–762.
Bolus injection protocol, 360, 726, 728,
731.
Bone, reflectance, 167.
Books on 3D LM, listing, 889.
939
Borohydride, to reduce glutaraldehyde
autofluorescence, 374, 770.
Botanical specimens, 414–439, 624,
784–785. See also, Plant cell
imaging, and Chapters 21 and 44.
birefringent structures, 420–421. See also,
Birefringence
deconvolution, 784–785
effect of fixation on, 195, 428
Equisetum, 774
fluorescence properties, 421–428
emission spectra, 421–423
microspectroscopy, 421–426
fluorescence resonance energy transfer,
425.
See FRET, 425
harmonic generation properties, 428,
711–715
light attenuation in plant tissue, 414–418
absorption spectrum, 415
A. thaliana example, 416
maize stem attenuation spectra, 417,
418
M. quadrifolia attenuation spectra, 416
M. quadrifolia optical sections, 419
Mie scattering, 162–163, 167, 417–418
nonlinear absorption in, 416–417
Rayleigh scattering, 162–163, 167, 417,
703
light-specimen interaction, 425–428
living plant cell, 429–439
calcofluor staining procedure, 424, 438
callus, 429
cell walls, 168–169, 188–189, 303,
306, 416–417, 420–421, 428–431,
435–136, 438, 439, 710–711,
713–715, 769–776, 779–781
chamber slides, use, 429
culture chamber, 429
cuticle, 434–437, 715, 717, 779
fungi, 438–439, 624, 782, 870
hairs, 431, 434–436, 772
meristem, 168, 420, 430, 770, 776–778,
783
microsporogenesis, 431–432
mineral deposits, 163, 420, 436–438,
703
pollen germination, 420, 433–434, 781,
783
pollen grains, 202, 305, 313, 420,
431–433, 553, 558, 781, 783
protoplasts, 195–196, 203, 416, 421,
423–427, 429–431, 438–439, 693
root, 172, 174, 303, 307, 421, 429,
430–431, 438, 464–465, 556,
772–773, 775, 777, 779–783
starch granules, 202, 420–421, 428,
432–433, 435, 703, 710–712, 715,
719
stem, 168, 172, 180, 417–419, 421,
424, 429, 556, 707, 710–711,
713–714
storage structures, 435–436
940
Index
Botanical specimens (cont.)
suspension-cultured cells, 189,
429–430
tapetum, 433–434, 779
waxes, 420, 428, 434–435, 714–715
point spread function in, 784
refractive index heterogeneity, 192,
418–420
maize stem, 419
Bovine embryo, 750.
Boyde, Alan, 2, 6, 141, 154, 224. See also,
Stereoscopic images.
BPNN. See Backpropagation neural
network.
Bragg grating, tuning diode, 107.
Brain slices, 392–398, 722–734. 686.
beam collimation, 728
choice of objectives, 395, 727–728
future directions, 929
image processing for, 732–734
algorithms, 733
alignment, center of mass in, 732–733
alignment, based on image overlap,
732
automatic detection of neurons,
733–734
drift/vibration compensation, 396, 732
image de-noising using wavelets, 734
image processing/analysis, 330–331,
395–396, 730–732
biocytin protocol, 730
classified using cluster analysis,
731–732
correlated electron microscopy, 731
montaging, 331
neuron reconstruction, 330–331, 730
protocol for PCA/CA, 731–732
spectral imaging, 382
two-photon/neurolucida system, 316
image production, 729
2-photon excitation, 727
deep imaging, 395
living neurons, 725
maintaining focus, 395, 732
microglia, 397–398
neuronal ensembles, 726
objective lenses, choice of, 727–728
second harmonic imaging, 729–730
in vivo observations, 387
preparation, 387
labeling cells, 394–396, 724–727
biolistic transfection, 724–725
bolus injection, 726
calistics, 726
choice of dyes, 729
diolistics, 726
dye injection/patch clamp, 726
genetic manipulation, 725–726
GFP transgenic mice, 726
Helios Gene Gun, 724
live-dead staining, 393
painting with AM-ester indicators,
726–737
photoactivation, 383
slice loading, 726
linear unmixing, 192, 382, 664–667
making brain slices, 393, 722–724
acute slices, 722–723
autofluorescence, 383
cultured slices, 724
mouse visual cortex, 723
primary visual cortex, 724
protocols, 731
thalamocortical slice, 724
photodamage, 729
pulse broadening, 728
reducing excitation light, 390–391
resolution, 729
second harmonic imaging (SHG),
729–730
silicon-intensified target (SIT) camera
use, 730
slice chamber, 394
protocol, 727
speckle microscopy, 383
useful techniques, table, 384–385
time-lapse, 382
two-photon imaging, 727
calcium imaging, 729
z-sectioning, 729
Breakdown.
electrical, in PMTs, 263, 660
optical, high power density, 198, 680,
682, 685, 687, 703, 705
Brewster surfaces, 83.
Brewster windows, 83, 102–103, 115.
Bright-field microscopy, 6, 127, 130, 201,
224, 229, 448, 468, 649, 728.
CCD for, 127, 483
deconvolution, 468, 472–473
depth of field, 4
low coherence light for, 130, 134–135,
139–140
optical projection tomography, 610–612
Brightness, source, 21, 26, 126–127,
129–130, 141–142, 215.
and exposure time, 141–142
gray levels, 71–73
as limitation of disk-scanners, 21, 215
of non-laser light sources, 126–127
of sun, 127, 135
Brillouin background, in glass fibers, 88.
Brillouin effect, reduction, 110.
Brownian motion, microtubules, 11.
BSL. See Backscattered light.
Buffering, fluorescent ion measurement,
740.
Bulk labeling, in living embryos, 761.
C
C. elegans, 746, 748, 766, 856, 857–858.
cryopreparation, 857–858
FRET imaging, 766
as model system, 746, 748
TEM images, 856, 857
Ca2+ imaging, see Calcium imaging.
Ca2+ indicators, 346–347, 738, 742–743.
See also, Ca2+ sparks, 737–738, 742.
discovery, 737, 738
Caenorhabditis elegans. see C. elegans.
Caged compounds, 759–760.
multi-photon excitation, 543–544
Calcein AM dye, 355, 360, 362–363,
426–427, 430, 685, 804, 812.
Calcium imaging, 529, 545, 584, 736–737,
812.
calibration, 742–743
data compression, 584
intensity image, 529
introduction, 736
multi-photon excitation, 545
ratiometric, 189
signal-to-noise ratio, 737
single-cell kinetic, 812
TIRF for measuring, 180
very fast imaging, 237
Calcium ion dyes, 183, 189, 237, 736, 737,
741–743. See also, fura-2, Fluo-3
and Indo-1.
Fluo-3 and Fura Red indicator system for
determining, 183
Fluo-3 indicator system for determining,
737
fura-2 reactions, 741–742
Indo-1 and Fura-2 indicator system for
Calcofluor, 424, 438.
staining procedure, 438
Calibration, 34, 75–76, 742–745.
Ca2+ sparks, 742
of CCD to measure ISF, 75–76
confocal microscopy, 742
errors in, 744
of ion concentrations, 742–745
ion interference, 745
of effective pinhole size, 34
in vitro, 742
Calistics, 726.
Callus, 429.
Cambridge Technology, galvanometers,
54.
cAMP indicators, 347.
Canna, 422, 710.
fluorescence spectra, 422
as function of excitation intensity, 165
nonlinear absorption, 710
Carbon arc lamps, 136.
CARS. See Coherent anti-stokes Raman
scattering.
CARS correlation spectroscopy (CS-CARS),
602.
Raman spectra, 602
CARV disk-scanning confocal microscope,
215, 226, 229, 230, 907–908.
diagram, 230, 907
CAT. See Computed axial tomography.
Cathode-ray tube (CRT), 5–6, 53, 67,
72–73, 291, 293, 588–589.
gamma, compensation, 73
Cavities, of dielectric coatings, 46, 47.
Index
Cavity-dumped lasers, 111, 114.
for FLIM imaging, 114
CCD. See Charge-coupled devices.
CD. See Compact disks.
cDNA-GFP fusion, in plants, 773.
Cedara, 281–282, 288, 302, 308.
Cell adhesion imaging with TIRF, 90.
Cell autofluorescence, 742.
Cell chambers, 11, 22, 191, 219, 370–371,
386–387, 394, 429–430, 564,
610–611.
for 4Pi confocal, 564
for biofilms, 870–873, 875, 877, 880, 885
brain slice, 394, 723, 727, 729
for epithelial cells, 370–371, 377, 386
finder chamber, 683
flow chamber, 870–873, 875, 877, 880,
885
for high-content screening, 810
for optical projection tomography,
610–611
perfusion, 394
for plant cells, 191, 429–430
simple, 22, 394
for SPIM, 613, 625, 673
table of required functions, 380
table of suppliers, 388–389
test chamber/dye, 654, 661
Cell cycle, 790, 791.
Cell damage, 2-photonmicroscopy, 680–688
See also, Bleaching; Photodamage.
absorption spectra of cellular absorbers,
681
intracellular chromosome dissection, 688
mitochondria, 686
nanosurgery, 219, 686–687
one-photon vs. multi-photon, 680–689
by optical breakdown, 198, 680, 682, 685,
687, 703, 705
photochemical, 682–685
absorbers/targets, 682
beam power sensor, 683
impact on reproduction, 686, 685
laser exposure parameters, 682–683
NIR-induced DNA strand breaks,
683–684
NIR-induced ROS formation, 683
photodynamic-induced, 684
spectral characteristics, table, 682
photothermal, 685
reproductive effect, short NIR pulses,
682, 686
ultrastructure modifications, 685–686
Cell microarray (CMA), 815–816.
Cell motility, 757.
Cell nuclei, optical effects, 23.
Cell pellet, three dimensional, 815.
Cell surface targeting assays, 813.
Cell walls of plants, 168–169, 188–189,
303, 306, 416–417, 420–421,
428–431, 435–436, 438, 439,
710–711, 713–715, 719, 769–776,
779–782.
labeling, 775
viability, 780
Cell-by-cell analysis, 817.
Cell-cell signaling, 778.
Cellular structures, optical effects, 22–23.
Center-of-mass alignment protocol, 733.
Center pivot/off-axis pivot mirrors, 1, 214.
Cerium, doping of quartz lamp envelope,
116.
CFP and YFP molecules, in FRET pair,
798–800.
Chambers for living cell imaging, 388–389.
commercial suppliers, table, 388–389
Charge amplifiers, 923–924.
defined, 923
destructive readout, 923
FET amplifier performance, 923
non-destructive (skipper), 923
Charge-coupled device (CCD), 26–28,
30–31, 39, 61–62, 65, 70, 74–78, 88,
127, 137, 142, 215, 233, 254,
458–459, 460–461, 482, 552, 558,
644, 754–755, 784, 918–931. See
also, Electron-multiplier
CCD.
bit depth, 75
camera, 918–931
advances in, for speed, 754–755
bright-field imaging, 127
for disk scanner systems, 78, 205, 215,
220, 233–235, 349, 459, 754–755
pixel size, 62, 65, 634–635, 784
specifications, table, 929
time for sampling, 70
choosing,
color, 927
computer-assisted pulse shaper, 88
confocal imaging, 458–459
cooled, advantages and limitations, 30–31
quantum efficiency, 26–28
spatial quantization of signal, 39
digital camera, 75
digital vs. video camera, 61–62
electron multiplier-CCD, 30–31, 76–77,
233–235, 262, 459–461, 482,
925–926
multiplicative noise, 77, 234, 257, 262,
926
result, 205, 234, 755
table, 233, 459
evaluating, 927–931
array size, 928–929
“the clincher,” 929
comparison, CCD/EM-CCD, table,
233, 459
dynamic range vs. pixel size, table, 928
maximum signal, 930
quantum efficiency, 927–928
readout noise, 928
readout speed, 928–929
self test, 930
sensitivity, 930
shutter stability, 929
941
specifications, 927, table, 233, 929
user-friendliness, 929
gain-register, 76–78, 460–461
intensified, 930–931. See also, Intensified
CCD
monitoring during exposure, 137
multi-focal multi-photon microscopy, 552,
558
noise sources, 256, 924–925
charge amplifier, 925
clock-induced charge (CIC), 234, 926
fixed pattern noise, 924–925
multiplicative noise, 77, 234, 257, 262
noise vs. pixel dwell time, 922
table, 256
operation, 254, 918–927
blooming, 923
charge amplifiers, 923–924
charge coupling, 918–920
charge loss, 921
dark charge, 921–922
destructive readout amplifiers, 924
edge effects, 921
electron multiplier, 926–927
FET amplifier performance, 253, 922,
924
frame transfer readout, 920
full-frame readout, 920
gain register amplifier, 925–926
incomplete charge transfer, 923
interline transfer readout, 920
leakage, 921–922
non-destructive (skipper) amplifiers,
923–924
possible problems, 920
quantum efficiency vs. wavelength,
922
quantum efficiency, 920–921
readout methods, 920
signal level representing zero photons,
925
storage array, 920
performance, table, 256, 459, 923
piezoelectric dithering, increases
resolution, 70
pixel size, 62, 65, 634–635, 784, 928
quantum efficiency and noise, 29, 644,
920, 922
measuring, 74–76, 926
sensors size, parallel data collection, 142
snapshot camera, 65
specifications, described, 927–930
testing, 930
Cheek cells, backscattered light image,
22–23.
Chemical environment probe, 517.
Chimeric fusion proteins, 794, 801–802.
anisotropy analysis, 794
cloning for FRET, 801–802
overexpression, 802
Chinese hamster ovary cell, 197, 556. 684+,
818.
Chirp, pre-compensation, 88, 111, 602, 907.
942
Index
Chlorophylls, autofluorescence, A. thaliana,
175, 194, 203, 425–426, 528, 711,
714, 779, 782, 881.
bleaching, 203
FLIM, 528
Cholera toxin transport, 790–791, 796–797,
802.
FRET, 796–797, 802–803
Chromatic aberrations, 134, 152–156, 178,
242–245, 657–658, 659.
apparatus for measuring, 243
axial chromatic registration, 243–345,
658, 657–659
of incandescent and arc lamps, 134
intentional, for color/height encoding, 154
lateral chromatic registration, 657–658
fluorescent latex bead labeled, 178
linear longitudinal chromatic dispersion,
154, 659, 664
measuring, 242–245
Chromatic corrections, 157, 177.
excitation/emission wavelength, 177
tube length, table, 157
Chromatic magnification difference. See
Lateral chromatic aberration.
Chromatin, 385, 390, 684, 693–695, 812.
Chromophores, 338–348, 543–544,
803–804. See also, Dyes;
Fluorophors; Fluorescent probes etc.
cellular introduction methods
electroporation, 359–360, 803
microinjection, 360–361, 388, 739,
748, 755, 795, 803–804
table, 344–345, 803
transfection reagents, 358, 360, 362,
556, 682, 790–791, 795, 803
multi-photon excitation, 543–544
CIC, clock-induced charge, EM-CCDs, 234,
926.
Circular exit pinhole, 9.
Circular laser beam, corrective optics, 106.
Classification, pattern. See Automated
interpretation of subcellular patterns.
Clathrin-GFP dynamics, 236.
Clearing agents. See also, Mounting media.
optical projection tomography, (OPT)
610, 624
plant material, 166, 417–420, 439,
774–775
Clock, role in digitizing and reconstructing
analog signal, 64.
Clock-induced charge, in EM-CCDs, 234,
926.
Closterium, 192–194.
chloroplast autofluorescence, 192–195
signal variation with depth, 194
CLSM. See Confocal laser-scanning
microscopy.
Cluster analysis (CA), 731–732, 826.
neurons classified using, 731–732
protocol with PCA and, 731–732
subcellular patterns, 826
CMA. See Cell microarray.
CNS, (central nervous system), 392–393,
395. See also, Chapters 19 and 41.
Codecs, image processing, 831, 836,
840–841.
Coefficient of variation, 660, 661.
Cohen’s k statistic, 826.
Coherence length, 7–8, 84.
defined, 7–8, 84, 130–131
reducing, for laser light, 84
Coherence surface, 84.
Coherence volume, 84.
Coherent anti-stokes Raman scattering
(CARS), 90, 204, 550, 595–605.
advantages, 204, 596
correlation spectroscopy, 602–603
defined, 595
energy diagram, 596
epi-detected, 597–599
forward/backward detected, 597–599
Hertzian dipole radiation pattern, 598
history, 595–596
imaging of biological samples, 603–604
adipocyte cells, 604
artificial myelin, 204
epithelial cells, 603
erythrocyte ghosts, 603
intensity distribution, 597
mapping intracellular water, 90
microscope schematic, 599
multiplex CARS microspectroscopy,
601–602
non-resonant background suppression,
600–601
energy diagram for multiplex CARS,
601
epi-detection, 600
phase control of excitation pulses, 600
picosecond vs. femtosecond pulses,
600
polarization-sensitive detection, 600
time-resolved CARS detection, 600
optimal laser sources, 599–600
pumped optical parametric oscillator
(OPO)
systems, 600
perspectives on, 604–605
unique features under tight-focusing,
596–597
Coherent illumination, 1, 83–84.
properties of laser light, 83–84
and resolution, 1
Collagen fibers, 164, 188, 313, 361, 393,
514, 703–704, 715.
autofluorescence, 545
birefringence, 164, 188, 717
gels, 393
polarization microscopy, 164, 188
second harmonic image (SHG), 703–704,
715
Collector optics, elliptical and parabolic,
129.
Colliding-pulse, mode-locked laser (CPM ),
540.
Colloidal gold labels, 167, 241, 846–859.
contrast, 167
electron microscope markers, 846–857
correlative, 850, 852, 855
SEM, 850
TEM
FluoroNanoGold, 854
GFP related, 854–855, 857–858
measuring resolution, 241
quenches fluorescence, 854
Rayleigh scattering, 167
Colocalization, 517, 650, 667–670, 794,
813, 881.
FRET, FRET, 519
erroneous, 581
Color display, 291, 292.
display space, 291
multiple channel display, 292
palette, 291
pseudo, 173–175, 190, 291
resolution, 291
true, 291
Color centers, in optics, avoidance, 116.
Color filters, 43–52. See also, Filters.
long-pass, 43–46, 175, 203–204, 212
short-pass, 45, 46
bandpass, 44, 45
Color print images, 592.
Color reassignment, 173–175, 190, 291.
Coma, 145, 151, 245, 247, 249, 483,
630.
distortion away from optical axis, 151
observation using point objects, 145,
246
Commelina communis, images, 712.
Commercial confocal light microscopes,
906–917.
BD-CARV II, 230, 907
La Vision-BioTec TriM-Scope, 907
Leica, TCS SP2 AOBS, 910
Leica MP RS Multi-photon, 910
Nikon C1si, 911
Olympus DSU, 913
Olympus Fluoview-1000, 912
optical parameters of current, table,
908–909
Visitech VT Infinity, 914
Visitech VT-eye, 914
Yokogawa CSU 22, 231, 915
Zeiss LSM 510 META optical, 916–917
Zeiss LSM-5-LIVE Fast Slit Scanner
schematic, 232, 916
Compact disks (CD) for data storage, 499,
586–587, 588, 731.
Compact flash cards, 588.
Components, of confocal fluorescence
microscopes, 43–58, 207–208.
acousto-optical devices, 54–57
chapter, 43–58
electroptical modulators, (Pockels cells),
25, 54, 57, 87, 116, 543, 701,
903–904
filters/beam-splitters, 44–51
Index
mechanical scanners (galvanometers),
51–54
polarizing elements, 58
Computed axial tomography (CAT),
610–611.
Compression, data see, Data compression.
Condenser lens, size, 129.
magnification, 128–129
Configuration of pixels in image plane, 62.
ConfMat. See Confusion matrix based
method.
Confocal disk-scanning microscope. See
also, Disk-scanning confocal
microscopy.
Confocal fluorescence microscope, 73, 207,
404–413. See also, Confocal
microscopy; Confocal laser-scanning
microscopy.
basic optical layout, 207
limitations due few photons, 73, 459
refractive index mismatch, 404–413 See
also, Refractive index
Confocal imaging, 4–5, 232, 235–236, 737,
738, 746–766, 809–817. See also,
next major head and
Chapters 35 and 36.
4Pi. See 4Pi microscopy
automated
for cytomics chapter, 809–817
of microarray slide, 816
platforms used for, 810
real-time, 810
temperature control, 810
types of assays for, 811, 813–814
workstations, 814
of biofilms, Chapter 50
deconvolution, 753. See Deconvolution
by disk-scanning confocals, 232
fast, 235–236
of fluo-3 loaded cardiac myocyte, 737
fluorescent indicators for, 738
high-resolution datasets, cell
arrangements, 776
of living cells, 813
of living embryos, chapter, 746–766
methods compared, 459, 644–647. See
Chapters, 22, 23, and 24
of plants, 773. See also, Chapters 21and
43
vs. non-confocal, 746
time-lapse. See Time-lapse imaging
Confocal laser-scanning microscopy
(CLSM), 9–15, 32, 38, 81, 89, 118,
222–224, 408, 518, 678, 690, 697,
750–751, 754, 884–885. See also,
next major head
advantages and limitations, 11–12,
222–223, 644–647, 884–885
alternatives to, 644–647, 754
comparisons, 644–647
disk-scanning and scanned slit, table, 224
digitizer employing full integration for, 32
edge response, 408
fluorescence lifetime imaging, chapter,
518
laser power required, 81
laser requirements for, 89
vs. multi-photon laser-scanning
microscopy, 750–751. See Chapters
22, 23, 24
photobleaching, 690, 697
vs. selective plane illumination
microscopy, (SPIM), 678
stage-or object-scanning, 13–15
TEM mode, 118
zoom magnification and number of pixels,
38
Confocal microscopy, 90, 141, 265,
381–399, 444–447, 453–467,
650–670, 742, 770, 774, 779, 810,
811, 815, 870–887. See also,
preceding major head and Chapters
35 and 36.
art of imaging by, 650
automated, platforms used for, 810
balancing multiple parameters for, 650
of biofilms, 870–887
calibration of, 742
cell microarray and, 815
colocalization, 667–670
effect of MLE and threshold, table, 669
fluorogram analysis, 669
image collection, 667–668
nerve fiber, 669
quantifying, 668
setting thresholds, 668
spatial deconvolution in studies,
668–670
vs. deconvolution, 644–647, 453–467. See
also, Chapters 22, 23, 24
CCD/confocal imaging combination,
458–459
deconvolving confocal data, 461–464,
466, 488–500
fluorescence excitation, 459
fluorescent light detection, 459–460
gain register CCDs, 460–461
image sections, figures, 455, 456, 462
imaging as convolution, 453–457
integration of fluorescence intensity,
459
limits to linearity, 457
model specimens, 461
noise, 459–463
out-of-focus light, 461
point spread function, 453–457
practical differences, 458, 463–466
resolution, 459–463
same specimen comparison, 465
sensitivity, 459–463
shift invariance, 457, 490, 564
single point imaged, 454
summary of pros/cons, table, 459
temporal resolution, 458
focus positioning, 651–652
getting a good confocal image, 629–631
943
alignment of optics, 629–630
back-focal plane (BFP), 210, 509, 629,
633
focus, 629
low signal, 631
mirror test specimen, 630
no signal, 631, 660
simultaneous BSL/fluorescence, 631
high-content screening systems, table, 811
illumination sources, 126–144, 650–651
See also, Lasers; Non-laser sources
acousto-optic tuning filter (AOTF),
651.
laser sources, chapter, 80–125
laser stability, 651
power measurement, 650–651
living cells, 381–399. See also, Living
cells
Minsky first confocal design, 2, 4–6, 11,
141, 216, 890
monitoring instrument performance,
650–663
illumination source, 650–651
optical performance, 652–660
photon efficiency, 14–15, 24
scan raster/focus positioning, 651–652
signal detection, 660–663
with non-laser light, 141
objective lens, 652–660. See Chapter 7
optical performance, 652–660. See also
Chapters 7, 11
axial chromatic registration, 658–659
axial resolution vs. pinhole, 656–657.
See also, Axial resolution
contrast transfer function, 656. See
CTF
coverslip thickness and RI, table, 654
field illumination, 658
flatness of field, 659
Focal Check™ beads, 657–659
lateral chromatic registration, 657–658
lateral resolution, 655
refractive index, 654. See Chapter 20
resolution test slides, 656
self-lensing artifacts, 659
spherical aberration, correction, 654,
655
subresolution beads, 655–656
x-y and z resolution using beads, 656
optimizing multi-labeling, 663–667
bleed-through between channels, 663
control samples, establishing limits,
663
measuring autofluorescence, 663
multi-tracking, reduces bleed-through,
664
positively labeled sample, 664
reflected light contribution, 663
secondary conjugate contribution, 664
photon efficiency, 24, 26, 28, 30 33–34,
36
polarizing elements, 57
scan raster, 651–652
944
Index
Confocal microscopy (cont.)
malfunctioning system, 653
phototoxicity from uneven scan speed,
651
sources of fluorescent beads, table, 653
well-calibrated system, 652
x and y galvanometers, 651–652
z-drive mechanism, 652
z-positioning calibration, 654
z-positioning stability, 652
separating signal by spectral regions for,
664
sequential collection reduces bleedtrough, 664
signal detection for, 660–663
coefficient of variation, 660–661
instrument dark noise, 660
PMT linearity, 661–662
signal-to-noise ratio, 660
spectral accuracy, 662
spectral detector systems, 662
spectral resolution, 662–663
wavelength response, 663
signal level, 444–445
signal-to-noise ratio, 444–447
spectral analysis, of plants, 770
spectral unmixing, 192, 382, 664–667
limitations to, 667
overlapping fluorophores separation,
664–667
removing autofluorescence, 667
stage-scanning, 9
staining plant cells, 774
vs. structured-illumination methods, 265
vs. two-photon excitation, 779
Confusion matrix based method (ConfMat),
826.
Constant output power laser stabilization,
86.
Continuous wave (CW) laser, 87, 88,
90–118.
beam intensity stabilization, 86–87
diode (semiconductor), 105–110
output power/cooling, 108
pumped solid-state, table, 94, 95
dye lasers, 86, 103, 112, 114, 124,
540–541
fiber up-conversion, 109–110
gas lasers
Argon-ion, 90, 102
Krypton-ion, 102
HeNe, 102
HeCd, 103
cesium and rubidium vapor, 103–105
table, 92–93
titanium-sapphire, 109
Contrast, 7, 11, 16, 37, 39, 49, 59–62, 68,
159, 162–204, 248, 421, 473, 488,
542, 599–600, 607, 622, 656, 657,
675. See also, Rose criterion and
CTF.
absorption, equations, 164
chapter, 162–206
defined, 162
flare, 649
formation of, chapter, 162–206
fluorescence. See Dyes, and Fluorophores
as function of feature size, 16,
61–62, 37, 634
intrinsic, 633
measuring, 16, 59
polarization. See Polarization microscopy
second harmonic generation. See SHG
and statistics, 633
third harmonic generation. See THG
Contrast medium, and laser power, 80–81.
Contrast method, defines signal required,
126.
Contrast transfer function (CTF), 16, 35,
37–39, 59–62, 656, 747.
in confocal vs. non-confocal microscopy,
16.
See Chapter 11
as function of grating period, 16
of microscope optical system, 35
relationship with objective BFP, 61
and spatial frequencies, 16, 37
and stages of imaging, 62
Control, of non-laser light sources, 138–139.
Convalaria majalis, 425, 556.
fluorescence microscopy of rhizome, 425
multi-focal multi-photon imaging, 556
Conversion techniques, 259–260.
analog-to-digital, 259
digital-to-analog, 259–260
Convolution, a primer, 485–487.
3D blurring function, 486
Fourier transforms, 487
geometrical optics, 487
out-of-focus light, 486–487
Cooling water, checking/maintaining,
116–117.
Cork microstructure, 770.
Correction collar, (spherical aberration), 15,
145–149, 158, 160–161, 178,
241–242, 247, 377, 407, 410–412,
471, 654–655, 657.
adjustment, 377, 407, 471, 499, 654–655
dry objectives, 410
multimedia, 640
Correctors, 70, 147.
spherical aberration, 15, 151, 147, 192,
411–412
Intelligent imaging innovations, 78–79,
151, 192, 395, 411, 654
to stored data, second Nyquist constraint,
70
Corrective optics, for diode lasers, 107–108.
Correlational light microscopy/electron
microscopy, 731, 434, 436–437,
846–860.
BSL image, 855
brain slices, 731then CLSM, 856–857
cryopreparation of C. elegans, 857–858
DIC image tracking, 849
DIC image/UV fluorescence image, 850
different requirement of LM/EM,
846–850
early 4D microscopy, 846
fluorescence/TEM to analyze
cytoskeleton, 854
fluorescent micrographs, 851
FluoroNanoGold for cryosections, 854
GFP, 854. See also, Green fluorescent
protein
HVEM stereo-pair, 848–849
immuno-stained bovine aorta, 852
LVSEM of FRAPed microtubules, 849,
850
phalloidin as correlative marker, 235–236,
344, 376, 378, 694, 696, 756, 804,
854–856
phase-contrast imaging, 851
postembedding, 855
quantum dot labeling, 853
same cell structure LM/SEM, 850–852
same cell structure LM/TEM, 852–856
SEM images at 5kV and 20kV, 847, 848
TEM cross-section of C. elegans, 856
TEM longitudinal section of C. elegans,
857
tetracysteine tag labeling, 221, 348, 357,
853
tiled montage TEM images, 858
time-series DIC images, 847
Correlative LM/EM. See Correlational light
microscopy/electron microscopy.
Coumarin dye, 114, 339, 344–345, 353, 355,
654–655, 661, 693.
Counting statistics, 20, 30. See Poisson
statistics.
Cover glass. See Coverslip.
Coverslip, and spherical aberration, 15,
147–150, 201. See also, Spherical
aberration.
CPM laser. See Colliding pulse mode-locked
laser.
Crane fly spermatocyte, metaphase spindle,
15.
Creep, in piezoelectric scanners, 57.
Cr:Fosterite, femtosecond pulsed laser, 109,
114, 415, 541, 706–709, 712–714.
Critical angle, for reflection of incident light
surface of refracting medium, 167,
502.
Critical illumination of the specimen,
128–129.
Crosslinking fixatives, 369.
Crosstalk.
between fluorescence channels, 203, 424,
882
between disk pinholes, 227
between excitation foci, 553–556,
558–559, 564
CRT. See Cathode-ray tube.
Crystal Fiber A/S, HC-800-01 bandgap
fiber, 88.
CSU. See Confocal scanning unit.
CTF. See Contrast transfer function.
Index
Curtains, laser, safety, 118, 904.
Cuticles, plant, 434–437, 715, 717, 779.
insect, 166
maize, 436
CW. See Lasers, continuous wave.
Cyan fluorescent protein (Cyan), 221–222.
Cyanine dyes, 339, 342, 344, 353–355,
362–363, 374, 443, 540, 587, 760,
854, 874.
Cytomics, 810, 811.
automated confocal imaging, 810
automated confocal imaging, table, 811
Cytoskeletal structures, 24, 188, 190,
328–329, 368, 370, 372, 378, 383,
461–462, 577, 703, 715, 719,
773–774, 813, 846–848, 852, 854.
LM-TEM analysis, 846, 854
stabilizing buffer, 852
Cytosolic markers, 757.
Cytotoxicity, reducing, 36–37. See also,
Bleaching; Phototoxicity.
D
DAC. See Digital-to-analog converter.
Damage threshold, LED sources, 139.
DAPI, 140, 344–345, 355, 358, 376, 431.
plants, 431
use of, 376
Dark current, 29, 76, 234.
fixed-pattern noise due to, 76
of photomultiplier tube, 29, 660
reducing, 234
Dark noise, defined, 232.
Darkfield microscopy, 5, 7, 172, 474, 672.
depth of field, 4
Data, 11–12, 33, 64, 76, 237.
conversion from ADU to electron data, 76
degradation by multiplicative noise and
digitization, 33
reconstructing, 64
speed of acquisition, 11–12
storage of volume of data, 237
Data collection guidelines, 319–320.
Data collection model, blind deconvolution,
472.
Data compression, 288–289, 292–293, 295,
319, 499, 580–585, 762, 764, 819,
835–836.
algorithms, 580
discrete cosine transform (DCT), 581
Huffman encoding, 580
Lempel-Ziv-Welch (LZW), 580
run-length encoding (RLE), 580
archiving systems, 580
gzip, 580
PKzip, 580
WinZip, 580
calcium imaging, 584
color images, 581
different techniques, table, 581
Dinophysis image, 585
effects on confocal image, 583
examples, 583–585, 592, 834–837
file formats for, 580–588
fractal compression, 581–582
GIF (graphics interchange format),
580
JPEG (Joint Photographic Experts
Group), 581–584
MPEG, 836–839, 840–841
PNG (portable network graphic), 581,
584
QuickTime, 829, 831, 836–837,
840–844
TIFF (tagged image file format), 580
wavelet compression, 581–584, 819
movies, 836–842
artifacts, 839
compression ratios, 842–843
entrope, 841
MPEG formats, 840–841
Up-sampling, 838
pixel intensity histograms, 584
testing, 830, 835
time required, table, 581for WWW use,
816
useful websites, 844–845
Data projectors, 590.
Data storage, 106. See also, Mass storage.
Data storage systems, 287, 395. 580, 594,
764.
chapter, 580–594
characteristics of 3D microscopical data,
287
databases, 861–869. See Databases
random access
CDR, CDRW, 586–587
DVD, 587
Magnetic disks, 586
semiconductor, FLASH memory, 588
for remote presentation, 842
role for STED, 577
Databases, 2D/3D biology images, 827,
861–869.
benefits, 863–864
fast, simple machine configuration, 863
improved analysis and access, 863
performance, 863
remote monitoring, 863
repeatability of experiments, 863
submissions to other databases, 863
criteria/requirements, 866–867
digital rights management, 867
metadata structure, 867
query by content, 866–867
user interface, 866
data/metadata management, 861–862
future prospects, 867
image database model, 864–865
image information management, 862
image management software, table, 865,
868
instrument database model, 864
laboratory information management
systems (LIMS), 862
microscopy data/metadata life cycle, 863
945
modern microscopes design aims,
862–865
projects, 865–866
BioImage, 865–866
Biomedical Image Library (BIL), 866
Scientific Image DataBase (SIDB), 866
recent developments, 861–862
MPEG-7 format, 862
relational database management
systems (RDBMS), 862
TIFF format, 861
software for, 868–869
ACDSee, 868
Aequitas, 868, 869
Cumulus, 868
Imatch, 868, 869
iView, 868, 869
Portfolio, 868
price, 868
Research Assistant, 868
ThumbsPlus, 868, 869
system requirements, 864
DBR. See Distributed Bragg reflector.
DCT. See Discrete cosine transform.
Deblurring algorithm, 476.
Deconvolution, 7, 26–28, 39, 40, 66,
189–190, 222–223, 278, 456–458,
464, 468, 488–500, 542, 564, 736,
746, 751–753, 778, 784–785, 828,
864, 900, 929. See also, Blind
deconvolution.
of 2-photon images, 498
and 3D Gaussian filtering, 70, 281, 285,
323, 392, 395, 667. See also,
Gaussian
4Pi lobe removal, 562, 565
advantages and limitations, 458, 475
algorithms, 472–476, 490, 495–497, 751,
778
comparison, 497–498
iterative constrained Tikhonov-Miller,
497
Jansson-van Cittert, 496
nearest neighbor, 495–496
non-linear constrained iterative,
496–497
Richardson-Lucy, 497, 568
Weiner filtering, 496
background history, 488–490
blurring process contributions, 488
equation showing restoration possible,
489
image formation, 489–490
schematic diagram of convolution, 489
blind, 189–190, 431, 463, 469, 472–473,
478, 486, 492, 496–497, 646
chapter, 468–487
maximum likelihood estimation,
472–477, 483, 669–670
blurring process contributions, 488
confocal data, 39, 40, 453–467, 488–500,
753, 778. See also, Confocal
microscopy, vs. deconvolution.
946
Index
Deconvolution (cont.)
of simulated confocal data, 40
CARS data cannot be deconvolved, 397,
399
chapter, 453–467
colocalization, 668–670 comparison of
methods, 66, 453, 467, 475–477,
497–499, 644–648
convolution primer, 485–478
convolution and imaging, 490–491
Fourier transform of PSF, 489, 490
linearity, 490
optical transfer function, 490–491
shift invariance, 457, 490, 564
and data compression, 584–585
test results, 401, 461, 464–466,
481–482, 483
defined, 189–190, 468
display of data, 301, 830, 835–836
examples, 40, 190, 392, 411, 462, 466,
471, 488–498, 510
4Pi, 468, 565
botanical specimens, 784–785
brightfield, 411, 475, 478
cardiac t-system, 498
confocal, 470
DIC, 470
polarized, 479
of simulated confocal data, 40
STED, 574–576
flatfielding the data, 477
black reference, 76
white-reference, 76
fluorescence lifetime imaging, 521
four dimensional deconvolution, 391–392,
752
Fourier transform of PSF, 489, 490
future directions, 483, 766
and image formation, 490–492
linearity and shift-invariance, 457, 564
live imaging, 480, 564, 751–754
missing cone problem, 494
model specimens, 461, 464–466,
481–482, 483
multi-photon, 488–500, 542
multi-view montaging, 330, 677
ion imaging, 736
noise, 495, 635
and Nyquist reconstruction, 59, 65, 67,
68, 222–223, 635
suppressing Poisson noise, 39
optical sectioning, 752
out-of-focus light, 26–28, 431, 487, 644
and pinhole, 26, 487
point-spread function (PSF), 223, 241,
247, 453, 463, 471, 489–492, 635,
655
approximations, 493
measuring PSF, 492–494
and Poisson noise reduction, 320
pre-filtering, 281, 497, 581
problem with specimen heterogeneity, 22,
648
purpose, 468
requirements and limitations, 489–494
diagram demonstrating convolution,
489
linearity, 490
missing cone problem, 494
noise, 495
optical transfer function, 490–491
point spread function, 489, 492–494
shift invariance, 457, 490, 564
sampling frequency, 635
spherical aberration, 471, 480–481
stain sparsity, 28
structured illumination, comparison,
265–279
subpixel refinement, 478–479
temporal/spatial, 392, 458, 753
transmitted light imaging, 472, 475,
478
of wavelength spectra, 382, 663–667,
771–772
limitations, 667
Deconvolution lite, 68–70.
Deflector, acousto-optical. See Acoustooptical deflector.
Defocusing, size and intensity distribution,
146.
Degree of modulation, 268–270.
locally calculated, 268–270
absolute magnitude computation,
268–269
equations, 269
homodyne detection scheme, 268–269
max/min measured intensity difference,
268
scaled subtraction approach, 269–270
square-law detection, 268–269
synthetic pinholes, 268
Delamination, and interference fringes,
168–170.
Delivery, dye, 355, 357–360, 810.
Deltavision, 132, 282.
Demagnification, and numerical aperture,
127.
Depth discrimination, in LSCM. See Axial
resolution.
Depth of field, 4, 9, 13.
extended-focus images, 9
fluorescence microscopy, 4
phase-dependent imaging, 13
Depth-weighting, projection images, 304,
306.
exponential, 304
linear or recursive, 304
Derived contrast (synthetic contrast),
188–201.
Descanned detection, 166, 208, 212, 537,
540–542, 754, 904.
Design of confocal microscopes, 43, 145,
166, 207–211, 237. See also,
Commercial confocal light
microscopes.
4Pi, 563, 566
of confocal fluorescence microscope, 208
efficiency in, 43
fast-scanning confocal instruments, 237
intermediate optical system, 207–209
of microscope optics, 145
MMM, 552, 555
practical requirements, 210–211
of transmitted confocal microscope, 166
Detection efficiency, 34, 35, 210–211.
measurement, 34–35
practical requirements, 210–211
Detection method, multi-photon, 541, 542.
descanned, 542
Detectors, 9, 11, 25, 28, 251–264. See also,
Photomultiplier tube; Chargecoupled device, etc.
area detectors. See Image detectors
assessment of devices, 260–262
CCD, 254
noise vs. pixel dwell time, 922
comparison, table, 255–256
conversion techniques, 259–260
descanned, 208, 212, 537, 540–542, 774,
904
direct effects, 252
errors, 211–212
evaluation, 211–217
future developments, 262–264
history, 262–264
image dissector, 254–255
image intensifier, 13, 232–233, 519–520,
524, 555–556
gated, 233, 519–522, 524, 555–556
intensified. See Intensified CCD
MCP-PMT. See Microchannel plate
microchannel plate, 232–233, 255, 262
MCP-CCD, 262
gated, 519, 523–524, 527, 532
noise internal, 256–259
internal detection, 256
noise currents, table, 256
photoemissive devices, 256–257
photon flux, 257–258
pixel value represented, 258–259
non-descanned, 185, 201, 218, 381, 447,
456, 507, 542, 552, 559, 643, 646,
727, 750, 779, 904, 909–910
phase-sensitive, 518–520, 619
photoconductivity effects, 252, 253
photoemissive, 254
photography. See Photographic systems
photomultiplier tube, 9, 11. See also,
PMT
photovoltaic effect, 252–253
photon interactions in, 252–256
point detectors, 260–261
quantal nature of light, 251–252
quantum efficiency (QE) vs. wavelength,
25
for second harmonic detection, table, 707
silicon-intensified target (SIT) vidicon,
730
spectral, 203–204, 662–663, 666–667
Index
TCPSC, 518, 520–523, 526
time-gated, 522
thermal effects, 252
work functions, table, 252–253
vacuum avalanche photodiode, 254, 255
Developmental biology, 545, 624.
multi-photon microscopy (MPM), 545
Dextran labeling, 173–174, 292, 512, 757.
DFB. See Distributed feedback.
4¢,6-diamidino-2-phenylindole, 140,
344–345, 355, 358, 376, 431. See
DAPI.
plants, 431
use of, 376
Diatom, 438, 638–640, 881.
as standard for measuring objectives, 145
test specimen, 638–640
DIC. See Differential interference contrast.
Dichroic filters, 212.
intensity loss, 212
transmission, 212
Dichroic mirrors (beam-splitters), 44, 45,
50–51, 129, 211, 217–218.
coating for collection mirrors, 129
double and triple, 217–218
effect of deflection angle, 211
separating emission/excitation, 44–45,
50–51
Die, of light-emitting diode, 133, 134.
Dielectric butterfly, galvo feedback, 54.
Differential interference contrast (DIC)
imaging, 10, 14, 76, 127, 146, 171,
453, 468, 473–475, 846.
blind deconvolution, 473–475
converting phase shifts to amplitude, 171
narrow bandpass filter use, 76
Nomarski DIC contrast, 2, 268, 746, 892
photon flux reduction, 127
schematic for, 475
three dimensional, 470
Wollaston prism, 156, 468, 473, 475
Diffraction, 61, 65.
contrast transfer function, 16, 35, 37–39,
59–62, 656, 747
and sharpness of recorded data, 65
Diffraction limit, 210–211. See also,
Rayleigh criterion.
defined, 210
point-spread function, 146
practical requirements for, 210–211
Digital light processor (DLP), projectors,
590.
Digital memory system, 64.
Digital microscopy, optics/statistics/
digitizing, 79.
Nyquist sampling, 146
Digital printers, 591–593.
Digital processing, in disk-scanning
confocal, 12.
Digital projectors, 590.
Digital rights management (DRM), 830,
844.
Digital video disks (DVD), 587–588.
Digital-to-analog converter (DAC), 64,
259–260.
operation, 64
Digitization, 25, 31–32, 36, 38–39, 59,
62–63, 66, 72, 75, 79, 259, 261, 286,
460, 495, 639, 911.
aliasing. See Aliasing
blind spots, 38
and Nyquist criterion, 38–39
precision, 25
and pixels, 62–63
of voltage output of photomultiplier rube,
31–32
DiI derivatives, 760.
Dimethylsulfoxide (DMSO), 697, 726–727,
760, 875.
handling, 739
DIN standard, microscopes, 156.
Dinophysis image, 585.
Diode injection lasers, 105–108.
Diode lasers, 86, 87, 107, 112, 116.
distributed feedback, 107
emission stability, 86
intensity, 87
maintenance, 116
modulated, 112
noise sources, 86
physical dimensions, 106
violet and deep blue, 107
visible and red, 107
wavelength stabilization, 87
Diode-pumped alkali lasers (DPAL),
103–105.
Diode-pumped lamp (DPL), 108–109.
Diode-pumped solid-state lasers (DPSS),
108–109, 111, 112.
kits, companies offering, 109
passively mode-locked, 111
ultrafast, 112
Diolistics, ballistic gene transfer, 726.
Dipping objective, 149. 161, 209, 411, 429,
568, 613, 727, 737, 870, 872.
Direct permeability, 358–359.
Discrete cosine transform (DCT), 581.
Disk-scanning confocal microscopy,
215–216, 224, 225, 228–229,
234–235 754, 755.
advantages and limitations, 223–224
for backscattered light imaging, 228–229
chapter, 221–238
commercial instruments, 907, 913, 915
comparing single- vs. multi-beam, 224
table, 226
and electron multiplier CCDs, 78, 205,
215, 220, 233–235, 349, 459,
754–755
embryo, 754
high-speed image acquisition, 216,
222–224, 754
image contrast in, 168–171
microscopes, table, 224
optical sectioning, 235
types, 228–232
947
Dispersion, optical, 56, 88, 152, 154, 242,
411, 542–543, 609, 683.
in acousto-optical devices, 3, 15, 55–56,
88
CARS signal generation, 728
compensation, 566–567
defined, 152
in fiber lasers, ultra-fast pulses, 88, 110,
113
by filter blank material, 211
generates third harmonic signal, 704–705
group delay dispersion, 537–538, 543
group velocity dispersion, 88, 111, 210,
537, 609, 903
in optical coherence tomography, (OCT),
609
in optical fibers, 502, 504, 507
and temperature, 15, 411
for multi-channel detection, 51
using to correct for chromatic aberration,
153
Display software. See Presentation software.
Displays, 580, 588–590, 594, 892.
cathode ray tube (CRT), 5–6, 53, 67,
72–73, 291, 293, 588–589
data projectors, 590
digital light processor (DLP), 590
halftoning vs. dithering, 589
international television standards, 589
liquid crystal (LCD), 589–590
supertwisted nematic (STN), 589
thin-film transistor (TFT), 589
monitors, 588–589
Distortion, 39–41, 152.
and resolution, practical, 39–41
Distributed Bragg reflector (DBR) diode
laser, 107.
Distributed feedback (DFB) diode laser,
107, 113.
ultrafast, 113
Dithering vs. halftoning display, 589.
DLP. See Digital light processor.
DMSO, 697, 726–727, 760, 875.
handling, 739
DNA damage, 390, 517, 539, 680, 682–684,
812.
DNA probes, 273, 317, 339, 343, 354, 358,
360, 362, 369, 393, 396, 459, 520,
531–532, 539–540, 691–695, 774,
779, 782, 812, 818–825, 828, 874.
DAPI, 140, 344–345, 355, 358, 376,
431
DRAQ5, 343
Hoechst, DNA dye, 136, 339, 344, 360,
362, 520, 565–566, 683, 782
DNA sequencing, constructs for, 801–802.
DNA transfer, 724, 756, 760, 773, 790,
802–804.
Dominant-negative effects, 755.
Donor/acceptor pair (FRET), 790, 792–794,
796–797. See also, FRET.
before bleach/after bleach ratio, 794
equations, 790, 792–794
948
Index
Donor/acceptor pair (FRET) (cont.)
fluorescence, 796–797
fluorophores, 794
separation in nm, table, 793
Double-image, diagram and example, 169.
Double-label, 375.
Down-conversion, parametric, 114.
DPAL. See Diode-pumped alkali lasers.
DPL. See Diode-pumped lamp.
DPSS. See Diode-pumped solid-state lasers.
Drift, 386–387, 652, 655, 732.
CCD read amplifier, 76
compensation for, 392–393, 732–733,
886
focus, 16, 40, 115, 190, 219, 386, 489,
567, 652, 720, 729, 886
compensating, 396, 732
lasers, 85–86, 115
DRM. See Digital rights management.
Drosophila, 273, 675–676, 747–748,
751–752, 754, 756, 759, 804, 810.
living embryo, 675–676, 752
salivary chromosomes, 273
SPIM, image, 675–676
Duty cycle, laser, defined, 110.
DVD, 587–588.
Dye lasers, 86, 103, 112, 114, 124, 540–541
in cancer treatment, 112
colliding-pulse mode-locked, 112
with intra-cavity absorbers, 112
noise and drift, 86
references, 124
as wavelength shifters, 103
Dye-filling, studying micro-cavities,
173–174.
Dyes, 22–23, 36, 44, 90–102, 109, 116, 118,
165, 173, 183, 212, 222, 342–346,
353–358, 360, 430, 461, 462, 527,
528, 575, 726, 736–738, 740–745,
748, 749, 755, 759–760, 774, 775,
782, 804. See also, Green fluorescent
protein (GFP); Rhodamine dyes;
Fluorescein.
affect on living cells, 391, 748
AlexaFluor, 353–355
Aniline Blue, 430–432, 435, 438, 774
APSS and Canna yellow, non-linearity,
165
bandwidth of emission, 44
BODIPY TR, methyl ester, 760
BOPIDY, 142, 342–343, 353–355, 389,
692, 749, 760–762
Calcein AM, 355, 360, 362–363,
426–427, 430, 685, 804, 812
calcium dyes, 346–347
cAMP, 347
characteristics of probes/specimen, table,
344–345, 354–355
coumarin, 114, 339, 344–345, 353, 355,
654–655, 661, 693
cyanine, 339, 342, 344, 354–355,
362–363, 374, 443, 540, 587, 760,
854, 874
diI derivatives, 355, 362, 389, 726, 760
donor acceptor pair, 794. See also, FRET
DNA probes, 343–344, 531–532,
818–825, See also, DNA probes
DRAQ5, 343
dyes vs. probes, 353, for embryos, 748,
761
exciting efficiently, 44
fade-resistant, 36 See also, Antifade;
Bleaching
for fatty acid, 347. See also, FM4–64,
below
Feulgen-stained DNA, 166, 200, 298,
433, 437
Fluo-3 and Fura Red, for calcium, 180,
183, 345, 434
Fluo-3 for calcium, 737
fluorescein, 353, 355. See Fluorescein;
FITC
fluorescence lifetime, 517, 527–528
FluoroNanoGold, 854
FM4–64, FM1–43, lipophilic dyes, 236,
355, 359–360, 389, 556, 755,
760–761
fura-2, 103, 189, 234, 257, 345, 346, 348,
358–359, 361, 529, 531, 726–727,
730, 733, 741–743, 810, 812, 846,
850
Fura Red, 180, 183, 345, 454
future developments, 348–349
genetically expressed, 348
Glutathione, 342, 358, 545, 694, 779,
782
hazards in using, 116, 118
for ion concentration, 346–347
ion-sensitive probes, table, 531
kinetics, 741–742
lanthanum chelates, 345–346
laser/filter configuration, table, 799
lineage tracers, 461
lipid dyes, 236, 355, 359–360, 389, 556,
755, 760–761
living cells, rapid assessment, table, 360
loading, uniformity, 749. See also,
Loading
LysoTracker Red DND-99, 360
membrane labels, 344–345
membrane potential, 205, 346
microinjection, 360–361, 388, 739, 748,
755, 795, 803–804
MitoTracker Red , 142, 170, 353, 358,
360, 430–431, 692, 750
multi-photon excitation, 543–544
nano-crystals, 343, 345. See also,
Quantum dots
Nile Red, 435, 528, 575, 774, 782
organic, 342–343, 353–356
oxygen sensor, 347
patch clamp loading, 360, 726, 734,
738–740
pH indicator, 346, 739–745. See also,
pH
imaging
photoactivatable, 187, 210, 224, 383, 385,
541, 544–545, 693, 729, 759–760,
912
Kaede, 187, 383, 385
Kindling, 574, 760
PA-GFP, 187, 383, 385, 752, 759–760
photodestruction, 340–341. See also,
Bleaching
and Chapter 39
photophysical problems, 338–340
absorption spectra, 339
autofluorescence, 339–340
contaminating background, 339–340
optimal intensity, 340
Rayleigh/Raman scattering, 339
singlet state saturation, 338–339. See
saturation, below
triplet state saturation, 339
phycobiliproteins, 338, 341, 343,
355–357, 693
for plants, 774–775. See Chapters 21 and
44
two-photon, 782
propidium iodide, 344, 355, 360, 426,
651, 693–695, 773, 778–779, 782,
812, 875, 877
quantum yield, 172, 180, 184, 338–845,
347, 353–354, 360, 363, 383, 421,
543–544, 574, 661, 683, 690–692,
710, 737, 792, 794–795
ratio methods, 346–348, 742–743
rhodamine, 353, 355. See also,
Rhodamine
excitation, 109
saturation , 21–22, 41, 142, 222, 265,
276, 338–340, 448, 643, 647, 899
Schiff-reagent, 262, 369, 770, 774–775,
778
selection criteria for, 353–358
signal optimization strategies for,
341–342
SNARF, 345–346, 531, 739, 744–745
specimen damage, 340–341
spectral properties, 212, 342, 344–345
spectral unmixing, 192, 382, 664–667
for STED, table, 575
Dynamic Image Analysis System (DIAS),
396–397, 783–784.
living cells of rodent brain, 396
of plant cells, 783–784
Dynamic range, 929–930.
E
e2v Technologies, EM-CCDs, 76–77,
233–234, 237, 262, 460, 925–926.
E-CARS. See Epi-detected CARS.
ECL. See Emitter-coupled logic.
Edge detector (software), 309, 322, 327,
396, 823–826.
Edge effect, self-shadowing, 172.
Edge-emitting diode laser, 89, 106.
corrective optics for, 89
cross-section through, 106
Index
Efficiency, laser, 102, 105–106. See also,
Quantum efficiency (QE); Photon
efficiency.
of diode injection lasers, 105–106
wall-plug, of argon-ion lasers, 102
EFIC. See Episcopic fluorescence image
capture.
EGS. See Ethylene glycol-bis-succinimidyl.
E-h. See Electron-hole.
Electro-magnetic interference, in electrooptical modulators, 57.
Electron microscopy, 167.
brain slices, 730–731
chapter, 846–860
cryo-techniques, 854
fixation, 167, 368–369
immuno-stained, 371–372, 852
micrographs, 479, 847–853, 855–858
tomography (EMT), 610–611
Electron-multiplier CCD (EM-CCD), 30–31,
74–75, 78, 142, 233–235, 262,
466–467, 482, 647, 678, 737,
753–754, 784, 923–926.
advantages and disadvantages, 30–31,
220, 228, 233–235, 237, 459–460,
647, 737, 909, 923–926
CIC, clock-induced charge, 234, 926
and disk-scanners, 76, 205, 215, 220,
270
frame-transfer, 262, 234
gain-register amplifier, 76–77, 258, 753,
925
interline-transfer, 233–234
mean-variance curves, 78
multiplicative noise, 77
noise currents, 256
parameters, vs. normal CCD, table, 233
QE(effective), 78, 927
readout amplifier, 76–77, 258, 753–754,
925
results, 235, 237, 755
Electron-beam-scanning television, 6–7.
Electron-hole (e-h) pairs and photon
counting, 29.
Electronic bandwidth, 64–65. See also,
Bandwidth.
Electronic noise, defined, 232.
Electronik Laser Systems GmbH, VersaDisc,
109.
Electrons, interaction with light, 129–130.
Electro-optical modulators (EOM), 25, 54,
57, 87, 116, 543, 701, 903–904.
Electroporation, 359–360, 795, 803.
for chromophores, 803
Ellis, Gordon, 2, 3, 7, 8, 13, 14, 84, 129,
131, 478, 507.
Embryo imaging. See Living embryo
imaging.
Embryos, 761–766.
bulk labeling, with dyes, 761
depiction, in time and space, 762–764
dyes, for multi-wavelength analysis, 756
FRET, 764–766
labeled proteins, 756
photobleaching, 759
transcriptional reporters, 756
EM-CCD. See Electron-multiplier CCD.
Emission filter. See Filters.
Emission spectra, of arc sources, 136, 176.
Emission spectra, fluorophores, 1- vs. 2photon excitation, 421.
Emitter-coupled logic (ECL), 259.
EMT. See Electron microscopy tomography.
Endomicroscopy, 511, 513, 514.
distal tip for, 514
fiber-optics, 513
human cervix image, 513
human gastrointestinal track image, 514
miniaturized scanning confocal, 511
Endoplasmic reticulum, 374, 770, 819.
and DiOC6, 390
FLIP, 382
FRET, 795
genetic fluorescent probes, 771, 783
and harmonic signal generation, 703
in ion-imaging, 738
and phototoxicity, 685
table, 363
Endpoint data analysis, 816–817.
Endpoint translocation/redistribution assays,
814.
Energy diagram, lasers, 102, 105, 106.
argon-ion laser, 102
helium-cadmium laser, 105
helium-neon laser, 105
semiconductor laser, 106
titanium:sapphire four-level vibronic
laser, 109
Energy, of single photon, 35, 127.
Energy transfer rate, for FRET, 790, 792.
Entrance aperture. See Back-focal plane.
EOM. See Electro-optical modulators.
Epi-detected CARS (E-CARS), 597–599.
erythrocyte ghosts, 603
Epi-fluorescence microscopy. See
Fluorescence microscopy, 44, 166,
172–173, 195, 202, 235.
Epi-illuminating confocal microscope, 9,
166. See also, Confocal laser
scanning microscopy; Confocal
microscopy.
Episcopic fluorescence image capture
(EFIC), 607–608.
mouse embryo image, 608
Epithelial cells, 14–15, 603.
CARS image, 603
oral, optical sections, surface ridges,
14–15
EPS. See Extracellular polymeric
substances.
Erythrocyte ghosts, CARS imaging, 603.
Ester-loading technique. See Acetoxymethyl
esters loading method.
Ethylene glycol-bis-succinimidyl (EGS),
369.
Euphorbia pulcherrima, spectrum, 710.
949
European Molecular Biology Laboratory
(EMBL), 53, 212.
compact confocal camera, 212
Evanescent waves, 90, 177, 180, 245, 503,
801.
defined, 90, 180
optical fibers, 503
resolution measurement, 245
Excess light. See Stray light.
Excimer lasers, 112, 116.
maintenance, 116
for tissue ablation, 112
Excitation efficiency, multi-focal multiphoton microscopy, 552.
Excitation filter, requirements, 44. See also,
Filters.
Excitation source, laser. See Lasers; Nonlaser sources.
Excitation wavelength change, contrast, 173.
Explants, for imaging living embryo,
748–749.
Exposure time, 62, 65, 71–76, 81, 127, 137,
141–142, 176, 212, 219, 224, 226,
231–236, 267, 270, 276, 346, 363,
392–393, 423, 427, 459–460, 477,
495, 556, 613, 627–628, 651, 655,
681–686, 692–697, 708, 746–747,
753–755, 760–764, 783–784, 816,
822, 850–851, 873.
for CCDs and EM-CCDs, 127, 137,
141–142, 231–236, 267
disk scanners, 231–235
laser, safety, 117–118, 839, 900, 903–904
reducing, 753–755
and source brightness, 141–142
total, comparison of methods, 442, 449
UV, 116
x-ray, 614–616
External laser optics, maintenance, 117.
External photoeffect. See Photoemissive
effect.
External Pockels cell, 25, 54, 87, 116, 543,
701, 903–904.
External-beam prism method, laser control,
90.
Extracellular polymeric substances (EPS),
183, 311, 358, 376, 703–704, 717,
760, 783, 870, 879–880. See also,
Collagen.
bleaching, 693
damage, 685
dye, 361
lectin-binding in biofilms, 870, 879–880
matrix, 760
negative contrast, 173
in optical projection tomography, 612
plants, 438, 783
preparation, 376
Extrinsic noise, reduction, 21.
F
Fabry-Perot interferometer, optical cavity,
81–82.
950
Index
Fast Fourier transform, 487.
to identify interference fringes, 202
Fast line scanner, 231–232.
Fatty acid indicator, 347.
FBG. See Fiber Bragg Grating.
FBR. See Fiber Bragg Reflector.
FBTC. See Fused biconical taper couplers.
F-CARS. See Forward-detected CARS.
FCS. See Fluorescence correlation
spectroscopy.
Feedback, 136, 139.
for control of light-emitting diode, 139
to increase source stability, 136
Femtosecond pulsed lasers. See Ultrafast
lasers.
Feulgen-staining, DNA, 166, 200, 298, 433,
437
Fianium-New Optics, Ltd., FemtoMaster1060
fiber laser, 113–114.
Fiber Bragg Grating (FBG), laser
stabilization, 87.
Fiber Bragg Reflector (FBR), stabilizes
laser, 87.
Fiber lasers, 85, 101, 109–110, 113–114,
124.
defined, 109–110
temperature sensitivity, 85
tutorial reference, 124
ultrafast, 101, 113–114
Fiber optics. See Chapter 26.
beam-splitters, 503–504
Bow-tie, pol-preserving fiber, 503
cable, for delivering ultrafast pulses, 88
laser output, 106
pigtail, 106
Fiber optics used in microscopy, 501–507.
evanescent waves in optical fibers, 503
fiber image transfer bundles, 504–505
fiber-optic beam-splitters, 503–504
fused biconical taper couplers, 503–504
glass made from gas, 501
gradient-index optical fibers, 501–502
key functions of fibers, 505–507
delivering light, 505–506
detection aperture, 506
diffuse illumination, 507
for femtosecond laser pulses, 507
large-area detection, 507
large-core fibers, as source/detection
apertures, 507
same fiber for source and detection,
506
single-mode fiber launch, 505
SMPP optical arrangement, 216
managing insertion losses, 506
angle polishing of fiber tips, 506
anti-reflection coating of fiber tips, 506
index matching of fiber tips, 506
microstructure fibers, 504
modes in optical fibers, 502
polarization effects in optical fibers, 503
polarization-maintaining fibers, 503
step-index vs. gradient index, 502
step-index optical fibers, 501–502
transmission losses in silica glass, 502
Fiber-optic confocal microscopy, 501–515,
893.
benchtop scanning microscopes, 507–508
clinical endomicroscopy, 513
distal tip, 514
human cervix image, 513
human gastrointestinal track image,
514
image transfer bundles, 504–505
managing insertion losses, 506
miniaturized scanning confocal, 508–512
bundle imagers for in vivo studies, 509
with coherent imaging bundles,
508–509
imaging heads, 508–512
objective lens systems, 509
optical efficiency, 509
optical schema, 508
resolution, 509
rigid endoscope, 511
vibrating lens and fiber, 510–511
in vivo imaging in animals, 510–514
Fiber-optic interferometer, 240–241, 504,
609.
diagram, 241
for measuring point spread function,
240–241
Fiber-optic light scrambler, 8, 13, 131–132,
143.
Fibroblasts, 292, 361, 691, 798, 803, 852.
Field diaphragm, 34–35, 127–128, 139, 461,
627, 648–649.
Field effect transistor (FET) CCD amplifier,
30–31, 77, 922–927, 929.
noise vs. pixel dwell time, 922
Filament-based lamps, 34, 44, 126–132,
135–138, 346, 507, 648, 663.
fiber optic, 507
image, 100 W halogen bulb, 135
size, 126–127
spectrum, 44, 136
stability, 34, 137
File formats, multi-dimensional images,
288–289.
Fill factor.
of CCD, 920–921, 927, 929
disk-scanning microscopes, 224–228, 233,
552
Filtering, digital, 281, 810. See also,
Deconvolution.
Gaussian, 41, 65. See also, Gaussian
filters
multi-dimensional microscopy display,
281
nonlinear, deconvolution, 190
sets, for automated confocal imaging, 810
smoothing, effect on contrast, 59
to reduce “noise” features, 70
Filters, optical, 43–51, 70, 89, 162, 190,
212, 753. See also, Heat filters.
conventional, 45
hard vs. soft coatings, 45–49
intensity loss, 212
interference, 45–51
conventional and hard coatings, 46
multi-channel detection, 51
ND filters, 43, 89
notch and edge, 50
tuning with angular dependence, 50
to select image contrast features, 162
short-pass, interference type, 46
transmission vs. laser line, 212
types, 46
wavelength selective, 43–51
FiRender, 281–282.
First or front intensity, projection rule, 302,
304.
FITC. See Fluorescein isothiocyanate.
Fixation, specimen, 368, 378, 428, 852, 854,
856.
antibody screening with glutaraldehyde
fix, 377
artifacts, 195, 369–373, 428, 624, 815,
854, 857
autofluorescence, 358, 663
borohydride to reduce autofluorescence,
374, 770
chapter 368–378
characteristics, 368–370
chemical fixatives, 369
crosslinking fixatives, 369
freeze substitution, 369, 769, 854–856
microwave fixation, 369
protein coagulation, 369
cryo-fixation, 854
dehydration, 166, 368, 417–418, 481,
611, 623–624, 815, 849, 854–855
effect on plants, 428
for electron microscopy, 167, 368–369,
372, 479, 731, 851–860
ethylene glycol-bis-succinimidyl, 369
evaluation, 371–374
cell height to measure shrinkage,
371–373
MDCK cell example, 372, 373
formaldehyde, 369–370, 373
general notes, 374–378
geometrical distortion, 372–373, 815
GFP, 854, See also, Green fluorescent
protein
arsenical derivatives, 348
glutaraldehyde, 369, 370
high-content screening, 815
immunofluorescence staining, 371, 372,
852
improper mounting, 376
microwave, 377–378
mounting methods, 370–374
critical evaluation, 371–374
media refractive index, table, 377
technique, 371
optical properties of plants, 428
pH shift/formaldehyde, 370–371, 373
Index
plants. See also, Botanical specimens,
Plant cells, 428, 769–770,
773–774
refractive index of mounting media, table,
377
optical effects, 428
refractive index of tissue/organs, table,
377
shrinkage, 369–373, 624, 815, 854
staining, 370–371
tissue preparation, 376
Fixed wavelength lasers, table, 119–120.
Fixed-pattern noise, 74–76, 278, 924, 927,
931.
Flare, out-of-focus light, 6, 132, 157–158,
172, 395, 456, 465–466, 469, 471,
481, 649, 731.
Flatness of field, 145, 151, 154, 418, 457,
639.
measurement/ small pinholes, 145, 457,
639
objectives, to improve, 151–152
Flat-fielding CCD data, 76, 477.
black reference, 76
white-reference, 76
Flexible scanning, 51–52.
FLIM. See Fluorescence lifetime imaging
microscopy.
FLIP. See Fluorescence loss in
photobleaching.
Flip mirrors, to control laser, 58.
Floppy disks, 586.
Fluorescein,48, 80–81, 88, 203, 261,
353–355, 375, 443, 582, 697, 781,
794, 930.
arsenical derivatives, 348
calculating laser power needed, 80–81,
443
derivatization, diagram, 354
double-labeling, 375
filters for, 48
photobleaching quantum yield, 363
rhodamine and, FRET between, 794
Fluorescein isothiocyanate, 88, 198, 203,
261, 263, 335, 375, 394, 397–398,
511–512, 527–528, 582–583,
693–694, 781, 794, 799, 884, 885.
See also, Fluorescein.
2-photon, 781
biofilms, 884–885
dextran, 292, 512
filter sets, 48–49
FRET, 794, 799
lifetime, 527–528, 532
photobleaching quantum yield, 363
toxicity, 391, 693–694
Fluorescence anisotropy measurements, 742.
Fluorescence contrast, 172–173.
Fluorescence correlation spectroscopy
(FCS), 5, 363, 383, 385, 602, 801,
803, 805, 917.
and CARS, 602
FRET, 801
laser requirements, 81
table, 385
Fluorescence emission, botanical specimens,
425–428.
1- vs. 2-photon excitation, 421
Fluorescence imaging, deconvolution vs.
confocal, 459–460, 644–648.
Fluorescence in situ hybridization (FISH),
316–317, 319, 323, 331, 333–334,
343, 875–878.
biofilms stains, 875–878
with fluorescent protein, 878
Fluorescence ion measurement, 736–738,
740–745. See also, Calcium imaging,
pH, etc.
calcium imaging, 736–737
concentration calibration, 742–745
indicator choice, 738
interpretation, 740–741
pH imaging, 346, 739–745
water-immersion objectives, 737
Fluorescence lifetime imaging microscopy
(FLIM), 108, 111, 114, 139, 204,
233, 382–383, 385, 516–533,
799–801.
advantages, 766, 800
alternatives to, 766
analysis, 251
applications, 516–518, 527–532
calcium imaging, 529
chemical environment probe, 517
FRET, 517–518
ion concentration, 517, 528–530
multi-labeling with dyes, 517, 527–528
pH imaging, 529–530
probes, 517
table, 530–532
comparison of methods, 523–527
acquisition time, 525–526
bleaching, 524
cost, 526
detector properties, 526–527
multi-exponential lifetime, 523–524
photon economy, 524–525
pile-up effect on detection efficiency,
526
shortest lifetime, 523
table, 526
decay process of excited molecule, table,
518
frequency domain methods, 518–520
disk-scanning implementations, 520
phase fluorometry method, 518–519
point-scanning implementations, 520
widefield, spinning-disk, 519–520
frequency-domain, 108
reducing repetition rate, 111
FRET, 799–801
history, 516
Jablonski diagram, 516, 517, 697, 792
with light-emitting diode sources, 139
limitations, 800
living cell images, 204
951
methods, 518–527
comparison, 523–527
frequency domain, 518–520
time domain, 520–523
multi-focal multi-photon microscopy,
555–556
quantitative fluorescence, 517–518
quantum efficiency, 516
spectroscopy, 516
table, 385
time domain detection methods, 520–523
point-scanning, 522
streak camera, 520
TCSPC FLIM, 522–523
time-gated FLIM, 523
use of intensified CCDs for, 233
Fluorescence loss in photobleaching (FLIP),
187, 382, 384, 801.
FRET, affected by, 801
table, 384
Fluorescence microscopy, 4, 9. 13, 43–44,
154, 166, 172–173, 195, 202, 235,
251, 448–451, 809–810 See also,
Widefield (WF) fluorescence
microscopy.
chromatic correction, 154
compared to disk-scanning microscopes,
235
vs. confocal imaging, 13
depth of field, 4
filters for selecting wavelengths for,
43–44
folded optical path, 166
increase contrast with less intensity,
172–173
signal-to-noise ratio comparative,
448–451
bleaching-limited performance,
448–450
configurations of microscope, 448, 449
disk-scanning microscope, 449
line illumination microscope, 449
saturation-limited performance, 450
scanning speed effects, 450–451
S/N ratios, table, 450
wide field (WF) microscope, 450
spectral problems, 44
Fluorescence, quenched by colloidal gold,
854.
Fluorescence recovery after photobleaching
(FRAP), 51, 54, 56, 90, 187, 210,
218, 224, 229, 237, 362, 382, 384,
390, 691, 759, 801, 805, 850.
in biofilms, 874
damage to cellular structure, 341,
859–851
damage to microtubules, 341, 850–851
efficiency of illumination light path, 210
related to TEM of same specimen,
850–851
setups for, 218, 907
table, 384
using CARV2 disk-scanner, 229, 907
952
Index
Fluorescence resonance energy transfer
(FRET), 26–28, 34, 184–187, 204,
218, 221–222, 382, 384, 425,
517–518, 556, 650, 691, 741–742,
764–766, 788–806, 796–797.
based on protein-protein interactions, 800
based sensors, 798–799
botanical specimens, 425
C. elegans, 766
chapter, 778–806
cloning and expression of fluorescent
constructs for, 801–804
donor/acceptor pair, 790, 792–794
donor, 796–797
efficiency, 792
experimental preparation, 795
FCS and, 801
FLIM and, 799–801
between fluorescein and rhodamine, 794
fluorescence lifetime imaging, 517–518
fluorescent proteins, 794–795
FRAP and, 801. See also, Fluorescence
recovery after photobleaching future
perspectives, 805
induced by cholera toxin transport, 797
intramolecular, 765
kinetics, 741–742
in living cells, 195–186, 204
chapter, 788–806
in living embryos, 764–766
MMM, 797–798
nanobioscopy of protein-protein
interactions
acceptor bleach for, 797–798
donor fluorescence for, 796–797
measurement methods for, 795
sensitized emission of acceptor,
795–796
photobleaching, 691
practical measurements, 792
probes, 221–222
quantum dots, 801
setups, 218
small molecules, 794–795
spatial orientation factor, 792–793
spectrofluorimetry, 793
spectroscopic properties used for, 795
standards for, 34
table, 384
theory, 790–794
TIRF and, 801
total, measured with widefield, 26–28
in transgenic animals, 765
wavelength depiction, 793
Fluorescence saturation, singlet-state, 21–22,
41, 142, 265, 276, 339, 448, 643,
647, 899.
Fluorescence speckle microscopy (FSM),
13, 383, 385, 889.
table, 385
Fluorescent biosensor, 799, 805.
future, 805
mitotic clock measurements, 799
Fluorescent constructs for FRET, 801–802.
cloning of fluorescent chimeras, 801–802
expression and over-expression, 802
functional activity of expressed, 802
Fluorescent dyes. See Dyes; Fluorescent
indicators; Fluorescent probes.
Fluorescent efficiency, 34.
Fluorescent emission, incoherence, 130.
Fluorescent indicators, 346–348, 736–743.
See also, Fluorescent probes, and
particular ions.
binding equation, 740–741
buffering, 740
calcium imaging, 736–737
calibration, 742–743
indicators, 738
cellular introduction, 738–739. See also,
Loading
cellular trapping, 738
choice, 738
concentration, 741–742
dialysis, 740
free diffusion, 741
genetically expressed intracellular, 348
green fluorescent protein, 348
ion indicators, 348
ligand-binding modules, 348
handling, 739–740
inaccurate measurements, 740–741
intracellular parameters imaged, 346–348
Ca2+, 346–347
cAMP, 347
fatty acid, 347
ion concentrations, 346–347
membrane potentials, 346
other ratioing forms, 347–348
oxygen, 347
pH, 346, 739–745
wavelength ratioing, 346
positive pressure, 740
selectivity, 743
Fluorescent intensity (IF), TIRF, 180.
Fluorescent labels, 342–346, 530–532, 761,
775. See also, Dyes; Fluorescent
probes; Chapters 16–17, and by
name of dye.
Fluorescent probes. 353–364, 387–389, 517,
530–532, 736–737, 739–740, 755,
769, 771, 773, 783, 806, 810, 811.
See also, Dyes, Fluorescence
indicators and by name of dye,
Chapters 16, 17.
automatic living cell assays, 811
bound, 737
care, 739–740
characteristics, table, 344–345, 354
development, 736
dye criteria for, 353–358
AlexaFluor dyes, 353–355
BOPIDY dyes, 353–355, 749, 760–762
coumarin dyes, 353, 355
cyanine dyes, 353, 374, 587, 760, 854,
874
dye classes, table, 355
dye vs. probes, 353
fluorescein, 353, 355. See also,
Fluorescein fluorescent proteins,
355–357
GFP, 355–357. See, Green fluorescent
protein indicators of intracellular
sate, 346–348
Ca2+ indicators, 346–347
protein
multi-photon excitation, 357–358
phycobiliproteins, 355–357
probes/specimen characteristics, table,
354
quantum dots, 357
rhodamine, 342–345. See also,
Rhodamine
excitation, 737, 344–345
for fluorescence lifetime imaging, 517,
530–532
genetically encoded, for plant imaging,
769, 771, 773, 783. See also,
Transcriptional reporters;
Transfection agents for high-content
screening, 810
high specificity/high sensitivity, 806
living cell imaging, 387–389
rapid assessment by, table, 360
loading methods, 358–360. See also,
Loading
acetoxymethyl esters, 359
ATP-gated cation channels, 359
ballistic microprojectile delivery, 360,
724–725, 802–803
direct permeability, 358–359
electroporation, 359–360, 795, 803
microinjection, 360–361, 388, 739,
748, 755, 795, 803–804
osmotic permeabilization, 359
peptide-mediated uptake, 359
transient permeabilization, 359
whole-cell patch pipet delivery, 360,
726–727, 734, 738–740
photoactivatable, 210, 224, 383, 385, 541,
544–545, 693, 759–760, 912
Kaede, 187, 383, 385
Kindling, 574, 760
PA-GFP, 187, 383, 385, 752, 759–760
photobleaching, 362–363. See also,
Bleaching
phototoxicity, 363–364 See also,
Phototoxicity factors influencing,
table, 363
specimen interactions, 361–362
cytotoxicity, 362
localization, 361–362
metabolism, 361–362
perturbation, 362
target abundance/autofluorescence,
360–361
tissues, 360
Fluorescent proteins, 187, 355–357, 739,
794–795.
Index
emission change after photodamage,
187
FRET, 794–795
genetically engineered variants, 739
ion binding regions, 739
Fluorescent lights, stray signal, 201, 632,
904.
Fluorescent staining, 371, 393, 438, 774.
See also, Dyes; Staining.
immunofluorescence, 371, 372, 852
living cells, 393
microglia, 319–320, 393–398
nuclei of living or dead cells, 393
Fluorite (CaF2), optical to reduce chromatic
aberration, 153.
FluoroNanoGold, cryosections, 854.
Fluorophores, 44, 338–349, 543–544,
664–667, 748, 794, 799. See also,
Dyes, Fluorescent labels.
Flying spot detector for measuring photon
efficiency, 34–35.
Flying spot ultraviolet (UV) microscope,
6–7.
Fly’s-eye lenses, for diode lasers, 107–108.
FM4-64, FM1-43, and other lipophilic
membrane dyes, 236, 355, 359, 360,
389, 556, 775, 760–761.
Focal CheckTM beads, 657–659.
Focal-plane array detection, 2-photon,
542.
Focal shift for mismatched RI, 405,
407–410, 553.
defined, 405
dependence, 410
for glycerol, table, 409
for water, table, 409
Focus, 3–4, 13, 36, 197.
for confocal microscope, 36
displacement, by living cell specimen,
22–23
effect of coverslip, 197
extended, 9
in phase-dependent imaging, 13–14
planes, diagram, 27
position, confocal microscopy, 651–652
Focused spot. See Point spread function.
Folded optics, for trans-illuminated confocal
microscopy, 166.
Formaldehyde, 369–370, 373–377, 428,
738.
AM-loading releases formaldehyde,
738
fixation protocol, 371
permeabilization agents for, 375
pH shift method, 370–371, 373
for plants, 428
stock solutions, 370–371
Förster distance, defined, 184, 790, 792,
793.
Förster equation, 184, 790, 793.
Förster resonance energy transfer. See also,
Fluorescence resonance energy
transfer.
Forsterite laser (Cr +4 in MgSiO4), 109,
114, 415, 541, 706, 707–709,
712–713.
second/third harmonic generation, 114
tunable, 109
Forward-detected CARS(F-CARS),
597–599, 603.
erythrocyte ghosts, 603
Foundations of confocal LM, chapter, 1–19.
Four-dimensional images, 746–749, 752,
761–764.
advantageous techniques, 746–747
automatic image analysis, 321
deconvolution, 495
embryogenesis visualization strategies,
761–764
living cells, 393
of living embryos
cellular viability, 747–748
challenges, 762
dataset display strategies, 393, 763–764
deconvolution, 752
for large thick specimen, 746–747
photobleaching during, 747–748
photodamage during, 746
required datasets for, 746–747
multi-photon, 535
structured illumination, 482
SPIM, 676
Fourier analysis.
4Pi microscope, 563, 576
analogy with image reconstruction, 69
of blind deconvolution, 472–476, 478
and convolution, 485–487
of image formation, 446, 454, 456–457
MRM, 618–620
of periodic test specimen, 638–639
of short laser pulses, 88, 728
SPIM multiview processing, 675–677
STED, 574
of structured-illumination images, 268,
270–273
and wavelet processing, 734
Fourier plane. See Back-focal plane, 201,
245, 509.
Fourier space, 270–271.
Fourier transform, 201, 202, 271, 487, 489,
490–492, 620.
of AC interference in image, 201–202,
651
and convolution, 487
and deconvolution, 487, 490–492
for detecting stray light into detector,
201
identifying interference fringes, 202
of microtubule TIRF image, 183
missing cone problem, 494
MRM image formation, 620
of point spread function, 489, 490
Fractal compression, 581–582.
Frame rate. See also, Speed
in confocal microscopy, 11
matching, 838–839
953
FRAP. See Fluorescence recovery after
photobleaching.
Free diffusion, of fluorescent indicators,
741.
Free-ion concentration, 742.
Freeze thawing, 731, 739.
Frequency, 52, 65, 82.
laser vs. pumping power, 82
of resonant galvanometer, 52
of sampling clock, 64
Frequency doubling. See Second harmonic
generation.
Frequency-resolved optical gating (FROG)
for pulse length measurement, 115.
FRET. See Fluorescence resonance energy
transfer.
Frustrated total internal reflection, defined,
177.
FSM. See Fluorescence speckle microscopy.
Full-well of CCD pixel, defined, 75.
Full-width half maximum (FWHM)
resolution.
4Pi, 562, 567
of beams in scanning disk, 554
of CARS, 597, 599
of confocal performance, 656–657,
661–662
of emission wavelength
LED, 136
quantum dots, 343
of interference filters, 44
laser bandwidth, 93, 95, 100, 101
laser pulse length, 109, 112, 507, 537,
538, 902
micro-surgery precision, 219, 687
multi-photon, 682–683, 901–902
objective resolution (PSF), 149, 209, 225,
444–445, 456, 492, 509, 552, 571
PMT rise time, 225
resolution, with spherical aberration,
407
table, 409
SPIM, 675
STED, 572, 576–578
z-resolution, measured, 194
Fundamental limits, chapter, 20–42.
Fungi, 438–439, 624, 782, 870.
Fura-2 [calcium ion] indicator dye, 103,
189, 234, 257, 345, 346, 348,
358–359, 361, 529, 531, 726–727,
730, 733, 741–743, 810, 812, 846,
850.
Fused bi-conical taper couplers (FBTC),
503–504.
Future, 143–144, 160, 192, 219–220, 234.
of EM-CCD with interline transfer, 234
of laser-scanning confocal microscopes,
219
of non-laser light sources, 143–144
spherical-aberration corrector, 15, 147,
151, 192
of tunable objective, 160
FWHM. See Full-width half maximum.
954
Index
G
Gain, 31, 232.
of image intensifier, 232
photomultiplier tube, from collisions at
first
dynode, diagram, 31
Gain register, (EM-CCD) 76–78,
233–234.
CCD (CCD), 76–78
of electron multiplier-CCD, 233–234
Gain setting, 75, 115.
defined, 75
effect of bandwidth on, 115
GAL4 genes, 773.
Gallium arsenide (GaAs).
diode laser, 107, 111
InGaAs photodiode, 707–708
LEDs, 133, 138, 143
PMT photocathode, 4, 28–29, 232, 252,
255, 263, 464, 527, 931
Galvanometer, 11, 25, 36, 40, 51–54, 56, 57,
63, 211, 215, 223, 231–232, 513,
543, 552, 558, 599, 651–652, 753,
806, 907, 910–911, 914, 931. See
also, Linear galvanometers.
defined, 52–54
distortion, 211
electromechanical properties, 40
errors, 40
in fiber-optic micro-confocal, 513
figure, 63
line-scanner, 231–232
linear, 52, 53, 223
measurement, 651–656
multi-focal, 554
multi-photon, 543
resonant, 25, 52–54, 56–57, 223, 447,
510, 539, 543, 552, 558, 910
specifications for, 214, 543
ultra-precise, 211
x-y scanners, 213–215, 223, 651–654,
806, 907, 910–911, 914
Gamma, brightness non-linearity, 72–73,
287, 832–833.
data projector, 590
display, 582–583, 589, 832–833
Gas lasers, 86, 90–105. See also, CW lasers;
Pulsed lasers.
continuous wave, 90–105
maintenance, 116
noise sources, 86
pressure, 102
Gating, intensified CCD, 25, 233, 262, 522,
555.
Gaussian beam profile, lasers, 80–81, 83–84,
108–109, 111, 113, 116, 231, 269,
338, 456, 496, 502, 538–539, 554,
891.
in CARS, 597
converted into line, 231, 916
fiber optic, 502, 505, 506
filling back-focal plane, 210, 509, 629,
633
“Gaussian-to-flat-wavefront” converter,
554
Kerr effect produces self-focusing, 111
laser beam profile, 538–539, 554, 597,
635–636
noise, 473, 497, 925
from optical fiber, 502, 505–506
optical tweezers, 89. See also, Laser
trapping spatial filter, 89, 729
Gaussian filters, digital, 39, 41, 65, 70, 89,
281, 285, 301, 323, 338, 391–392,
399, 497, 499, 510, 650, 667–668,
676, 729, 734, 753, 764, 830.
of 3D data to reduce Poisson noise, 39,
41, 65, 69–70, 269, 281, 285, 323,
391–392, 399, 499, 510, 635–636,
650, 667–668, 676, 764, 830
“Gaussian blob,” 635–636
and Nyquist reconstruction, 65
in presentation displays, 830
results, 285, 676, 733, 835–837
Gaussian laser pulses, 536–536, 902.
Gaussian noise, 473, 497, 925.
Gaussian norm statistical tests, 830, 835,
837.
GDD. See Group delay dispersion.
Gene gun, 360, 724–724, 730.
Geometric contrast, 180–187.
Geometric distortion, 6, 23, 36, 39–41, 53,
152, 211, 215–216, 265, 297, 329,
372–373, 448, 480, 590, 641,
653–654, 741, 835.
kinetic, 741
measurement, 651–656
projector, 590
of specimen preparation, 372–373, 815,
872
Gerchberg-Saxton algorithm, deconvolution,
472.
GFP. See Green fluorescent protein.
Ghost images, from transmission
illuminator, 201–202.
GIF (Graphics interchange format), 580.
Gires-Tournois interferometer (GTI), to
reduce GVD, 88.
Glan-Taylor polarizer, 85, 87, 100, 171.
in single-sided confocal microscope,
171
Glan-Thompson polarizer, attenuator, 85,
904.
Glutaraldehyde, fixative, 369, 369–374,
377–378, 428, 438, 731, 852.
antibody screening with, 377
autofluorescence of, 374, 428, 770
fixation protocol, 370
stock solutions for, 370
Glutathione (GSH), 342, 358, 545, 694, 779.
visualization, in plant cells, 782
Glycerol, immersion/mounting medium,
404, 407, 409–410, 435, 563, 654,
698, 785.
clearing, 198, 200
diffusion in, 698
immersion objective lenses, 412, 563, 567
example, 785
mounting media, 371, 373, 375, 377–378,
420
RI-mismatch, table, 409, 410
Goggles, laser, for eye protection, 118.
Gold’s ratio method, 476.
Golgi receptor, 374, 389, 556, 564–566,
791.
Golgi stain, 107, 283, 298.
Gourard shading, 308, 309, 311.
Gouy phase shift, 597.
Graded index (GRIN) lenses, 84.
in diode lasers, 108
Gradient index optical fibers, 501–502.
Gradient-weighted distance transform,
323.
Graphics interchange format. See GIF.
Grating, periodic.
GVD compensator, 88, 504, 538, 686
laser tuning, 90, 103, 106–107, 111
minimum spacing, 1, 16, 652
OCT phase-delay, 609
pulse compressor, 113
spectral detector, 87, 346, 422, 664,
772
structured illumination, 266–267, 273
Gray levels, 71–76.
intensity spread function, 74–76
printer, 592
Green fluorescent protein (GFP), 90, 174,
221–222, 348, 355–357, 429,
478–479, 556, 568, 571, 612, 614,
625, 675–676, 690, 692, 698–699,
724–725, 727, 731, 741, 747–752,
755, 756–763, 766, 769–773,
781–785, 798–806, 812–815, 820,
854–859, 862, 873–875, 877–879,
885. See also, Transfection reagents;
Transcriptional reporters.
biofilms labeling, 873
or CFP molecules, as FRET pair, 798
constructs, in embryos, 756
EM imaging, brain cells, 731, 854–859
FRET, 793–795, 798–803
image contrast, 174
limitations, 760
membrane localized, 749
methods with Correlative LM/EM, 854
mice, 727
photoactivatable, 187, 383, 385, 752,
759–760
photobleaching, 690, 692, 698
for plant imaging, 424, 429–430,
769–773, 781–785
direct visualization, 773
genetic fusions, 773, 783
genetic marking, 773
two-photon excitation, 782–783
protein fusions/cytoskeleton, 773–774,
801
tagged proteins, 758
TIRF, 90
Index
FRET, 794
Grey levels, 71–76.
printer, 592
GRIN. See Graded index.
Ground state depletion (GSD), 573.
Group delay dispersion (GDD), 537–538,
543.
Group velocity dispersion (GVD), 88, 111,
210, 537, 606, 609, 903.
in optical coherence tomography, 609
pulse broadening due to, 88, 111, 210,
537–538, 543, 606, 609, 728,
903
GSD. See Ground state depletion.
GTI. See Gires-Tournois interferometer.
Guinea-pig bladder, calcium sparks, image,
237.
GVD. See Group velocity dispersion.
Gzip, 580.
H
Hairs, plant, 431, 434–436, 772.
Halftoning vs. dithering, 589.
Halogen lamps, 126–127, 132, 136–139,
143, 159, 663.
brightness vs. temperature, 136
filaments, 132
image, 135
lifespan, 136
power available, 126–127
stability plot, 137
Haralick features, 818–820.
Hard coatings, for interference filters, 45,
48.
Hard copy, 580, 590–594.
photographic systems for, 590–591
printers, 591–593
aliasing, 592
color images, 592
digital, 591–593
grey levels, 592
ink jet, 593
laser, 593
posterizing, 591
scaling techniques, 592
Harmonic signals, 2, 49, 80, 90, 100, 109,
113–114, 162–163, 174, 179–180,
188, 243, 361, 414, 428, 535, 545,
550, 556, 577, 596–597, 682,
703–704, 708–719, 722, 729, 734,
894 See also, Second harmonic
generation; Third harmonic
generation see Structured
illumination.
chapter, 703–721
contrast, 179–180, 188
descanned detection, 56
in lasers, 109, 113, 114, 115
plants, 428
second and higher, 114
Haze, from out-of-focus light, 227.
HBO-50 mercury-arc bulb, 126.
HCS. See High content screening.
Heat, 84–85, 89–90, 109, 129, 133.
filtering, dichroic filters, 43–44, 129,
132
heat sink for LED light source, 133
from laser cooling, 84–85, 109
of optical trap, 89–90
placing system components, 129
Heat filters, to exclude IR light, 43–44, 129,
132.
liquid, 132
Heating. See also, Thermal variables.
detectors, 252
microwave fixation, 377
in magnetic resonance imaging, 621–622
multi-focal, multi-photon, 551, 556, 685,
903
specimen, by the chamber, 387–389, 394,
732
specimen, by the illumination, 43, 89,
132, 211, 218, 341, 536, 539, 544,
556, 621–622, 681, 685, 884, 903
calculation, 89, 685, 904
stability, 652
HeLa cells, 391–392, 693, 799, 812, 814,
820, 828, 854.
Helios Gene Gun System, 724.
Helium-cadmium (He-Cd) laser, 83, 86, 90,
93, 103, 105, 115.
operational lifetime, 115
output variation, 86
transverse electromagnetic mode, 83
Helium-neon (He-Ne) laser, 82, 84, 88–90,
93, 102–103, 105, 107, 240, 241,
376, 673, 680, 798, 799, 864, 875.
four state, 82, 105
Heterectis crispa, 874.
Hidden-object removal, 304–305.
High content screening (HCS), 809–817.
for cytomics chapter, 809–817
data management/image informatics,
816–817
fluorescence analysis of cells, table, 812
multiple fluorescent probes, 810
High resolution spatial discrimination, 813.
High throughput screening (HTS), 809.
High voltage electron microscope (HVEM),
846.
stereo images of platelets, 848–849
Hippocampal brain slices, 268, 316–317,
393, 556–557, 722, 724–725, 727.
calcium imaging, 556–557
culture protocol, 724–725
damage, 341
at neurons, 205, 268, 316–317, 393
Histology, 623, 624.
Historic overview of biological LM, table,
2–3.
Hoechst, DNA dye, 136, 339, 344, 360, 362,
520, 565–566, 683, 782, 812.
4Pi, image, 565–566
FLIM image, 521
high-content screening, 812, 814
Holey optical fiber/non-linear effects, 88.
955
Holographic diffusers, to reduce coherence,
84.
Holography, holomicrography, 7–8.
Hooke, Robert, image of cork, 769–770,
785.
HTS. See High throughput screening.
Huffman encoding, 580–581.
Human endomicroscopy, confocal.
cervix, 513
gastrointestinal track, 514
Human retina, viewed with OCT, 609.
Huygens, 3D software, 104, 413, 669, 778.
Huygens-Fresnel wavefront construction,
406.
HVEM. See High-voltage electron
microscope.
Hybrid mode-locked dye laser, 540–541.
Hymenocallis speciosa, fluorescence spectra,
422.
Hysteresis.
in Piezoelectric scanners, 57, 754
temperature cycling of lenses, 249
I
I5M, (Incoherent Illumination Image
Interference Imaging), 275, 561,
569–570, 672.
optical transfer function (OTF), 569–570
ICNIRP. See International Commission of
Non-Ionizing Radiation Protection.
ICTM. See Iterative constrained TikhonovMiller algorithm.
IEC. See International Electrotechnical
Commission.
IF. See Fluorescent intensity.
Illumination, 44, 210. See also, Structuredillumination microscopy, and
Chapter 6.
brightness, table, 140
errors, 211–212
evaluating, 211–217
goal in confocal microscopy, 210
path, 211–212
types of lamps, 44
vignetting caused by beam shift, 211–212
Image(s), 9, 11–12, 30–31, 38–39, 59, 145,
192, 210, 219, 280, 286–290. See
also, Multidimensional microscopy
images.
contrast, 7, 11, 16, 39, 49, 60–62, 68,
159, 162, 165, 167, 173–175, 180,
189–190, 192, 201–204, 248, 421,
473, 488, 542, 599–600, 607, 622,
656, 657, 675
chapter, 162
flare, 649
definition, 280
degradation of, measuring, 145
extended-focus, 9
motion between specimen and objective,
39
multi-dimensional microscopy, 286–290
anisotropic sampling, 287
956
Index
Image(s) (cont.)
calibrating image data, 286–288
contrast transfer function (CTF), 61.
See CTF
data type/precision in computations,
288–289
digitization, defined, 62
dimensions, 286–288
display devices, non-linearity of, 72–73
file formats, table, 288–289
processor performance, 289–290
Voxel rendering speed, 290
real, disk- and line-scanners, 30–31
reconstructing, and noise reduction,
38–39. See also, Reconstruction;
Nyquist reconstruction sharpness of
vs. signal intensity, 192
of source and detector pinholes, 210
speed of acquisition, 11–12. See also,
Speed as sum of point images, 59
thermal distortion, 219. See also, Thermal
variables
Image analysis. See Automated 3D image
analysis methods; Automated
interpretation of subcellular location
pattern.
Image dissector, 254–255.
in trans-illumination mode, 10
Image enhancement. See Deconvolution,
488–499.
Image iconoscope, for television, 6–7.
Image intensifiers, 13, 232–233, 235, 255,
460, 477, 519–520, 522, 524,
555–556, 730, 737, 784, 801, 930.
Image Pro Plus, 282, 290.
Image processing. See also, Automated 3D
analysis methods, and Multidimensional microscopy display.
for display, Chapter 14
for measurement, Chapter 15
Image resolution, 8, 9. See also, Resolution.
Image substrate, automated confocal, 810.
ImageJ, free software, 282, 290, 395,
732–733, 762–764, 795, 858.
Imaging system, optics characterized by
CTF, 61.
Imaging techniques, 382–386, 394–395.
combining fluorescence with other,
383–386
fluorescence correlation spectroscopy,
383
fluorescence lifetime (FLIM), 382,
516–532
fluorescence loss in photobleaching
(FLIP), 382
fluorescence recovery after
photobleaching, 382
fluorescence resonance energy transfer,
382
fluorescence speckle microscopy (FSM),
383
laser trapping, 383
linear unmixing, 192, 382, 664–667
multi-channel time-lapse fluorescence,
382
optical tweezers, 383
photoactivation, 187, 224, 383, 385, 541,
544–545, 693, 759
photo-uncaging, 383. See also, Photouncaging physiological fluorescence,
383
spectral, 382
table, 384–385
time-lapse fluorescence, 382
Imaris, software, 193, 281–282, 284,
287–288, 290–291, 299, 301–303,
308, 311–312, 764, 795.
In vitro fertilization, mitotic apparatus, 188.
In vitro preparations.
2D mixed-cell, assays, 813
antifade agents. See also, Antifade, 342
automated analysis, 318–320
backscattered light image, 513
biofilms, 870, 872, 879, 884
bleaching, 551, 851
brain slices. See Brain slices, 392–393,
725
cell maintenance, 387
cytoskeleton, 368
fertilization, 188
GFP, 357
high content screening, 809, 813–816
high speed imaging, 11, 237, 809, 813,
815–816
ion imaging, calibration, 742
living cell imaging, 387
micro-CT, 614, 617
micro-MRI, 618, 621, 623–625
multi-photon, 535
optical coherence tomography image, 609
photodamage, 684
In vivo (intact animal) imaging, 112,
368–377, 512, 545, 806.
2-photon microscopy (MPM), 535, 543,
545
cell preparations, 387
comparison with fixed material, 368–377
FLIM calibration, 517
labeling, 372–373
miniaturized confocal, 504, 508, 511–513
micro-CT, 614, 617
micro MRI, 618, 621, 623–625
molecular imaging, 806
photodamage, 684, 693–694, 698
“stick” lenses, 806
Incandescent lamps, 34, 126, 133–137, 477,
499 See also, Halogen lamps.
black-body radiation emitted by, 135–136
spectrum vs. temperature, 137
stability, 137, 477
Incidence angle, 49, 50.
efficiency, 143
interference filters/transmission, 49
reflectivity, diagram, 50
Incident light beam, sample interaction,
162–163.
Indo-1, calcium indicator, 103, 189, 257,
345, 346, 348, 529, 531, 544, 693,
697, 742–743.
Infinity corrected optics, 155–157, 166, 239,
405.
advantages, 156–157, 166, 239, 405
Infinity PhotoOptical, InFocus spherical
aberration corrector, 15, 151.
Infinity space, generating, 157.
Information, 27, 60, 64, 73–74, 179, 235,
241, 243, 268, 270–275, 278, 330,
334, 353, 369, 382–383, 396, 398,
443, 448, 459, 468, 475–476, 481,
487, 488–490, 494, 496–499, 506,
512–513, 517, 519, 522–524,
543–544, 556, 559, 570, 580–587,
596, 643, 650, 732, 715, 769, 774,
776, 779, 782, 790, 794, 800.
3-dimensional, 321, 378, 396, 747
4Pi, 570
and bleaching, 222, 690–692, 705
CARS, 597–598, 602
colocalization, 668
confocal, 461, 462
contrast, see Chapter 8 and Contrast
crystal orientation, 179, 188
display of, 280–281, 288–291, 293,
295–297, 299–301, 304–305, 311
efficiency, 336, 628, 631
of electronic signal, limitations on, 64
genetic, 756, 762–763
lost signal, 25–28
matching gray levels to, 73–74
micro-CT, 615
micro MRI, 618
and Nyquist sampling. See Nyquist
sampling, 38, 39, 634–637
chapter, 59–79
optical projection tomography, 612
out-of-focus light, 27, 368, 458, 461, 746,
784
parallel vs. serial acquisition, 223–224
PSF, 245, 247, 250
from second harmonic generation signal,
179
Shannon theory, 443
on source brightness, 137
spectral, 665–667
SPIM, 614, 675–378
storage, 106
chapter, 580–594
theory, 4, 64, 443
transmission, contrast transfer function,
37, 60
Index mismatch. See Spherical aberration.
Infrared (IR) lasers, 89, 383, 385. See also,
Ultrashort lasers; Laser tweezers.
solid state lasers, 108–109
Infrared paper, to identify infrared beams for
safety purposes, 118.
Ink jet printers, 593.
Innova Sabre/frequency-doubling crystal,
102.
Index
Insect cuticle, transparency to NIR light,
166.
Installation requirements, for laser sources,
85.
Instrument dark noise, 660. See also, Noise
Integrated circuit (IC) chip, 9.
Intelligent imaging innovations, (III), 3D
imaging system supplier, 78–79, 151,
192, 395, 411, 654.
Intensified CCD, 13, 232–233, 460, 477,
519–522, 524, 555, 556, 737, 784,
930.
Intensity, light, 26, 37, 43, 58, 59, 61,
71–72, 86, 87, 133, 136, 163, 165,
180, 189, 192, 208, 217, 222, 228,
258, 270, 391, 413, 426, 459, 461,
487, 536, 538, 571–573, 633, 681,
693, 705, 810, 901.
of excitation light, 80, 222, 680–682
laser beam, stability, 86
losses
detection path, table, 217
illumination path, table, 217
minimum needed, 392
on optical response of specimen, 165
in photons/second, 80
regulating, 43, 88
singlet-state saturation, See Saturation
and visibility, 37
Intensity control.
continuous wave laser, 88
non-laser, 128
Intensity distribution, 146–154.
of Airy disk, 65, 146. See also, Airy disks
changes with focus, 147, 407, 455,
463, 471
effect of coverslip thickness, 149
effect of RI mismatch, 148. See also,
Spherical aberration
in focal spot, plots, 147–154
nonsymmetrical change with focus,
148
unit image, 147
with astigmatism, 152
with coma present, 151
with spherical aberration, 148–150,
212
Intensity loss, with spherical aberration in
detection path, 148–150, 212.
See Spherical aberration.
Intensity spread function (ISF), 74–78.
CCDs and PMTs compared, table, 78
defined, 75
estimating intensity measurement error,
76
and gray levels, 74–75
measuring, 75
Interference contrast.
differential interference contrast, (DIC),
10, 14, 76, 127, 146, 171, 453, 468,
473–475, 846, See also, Differential
interference contrast.
deconvolution of, 473–475
phase-contrast, 9, 171, 368, 372, 453,
506, 643, 649, 731, 851, 854, 890,
892. See also, Phase contrast
centering the phase rings, 643. See
also, Bertrand lens scanning, 9, 13
using fiber optics, 506
RI inhomomogeneity and contrast, 22–23,
41
Interference filters, 45–51, 102, 136, 212.
in argon-ion laser systems, 102
continuously-graded, 137
destructive and constructive reflections,
45
transmission, 212
types, 46–49
Interference fringes, coverslip surface, 168,
170.
Amoeba plasma membrane/coverslip, 170
in close proximity, 168
Interference mirrors, 46.
Interference mode, coherent light, 130.
Interference, speckle pattern, 8, 13, 84, 90,
130–132, 144.
in backscattered light images, 448
fluorescence speckle microscopy (FSM),
13, 383, 385, 889
Interferometer.
4Pi microscopy, 561
Fabry-Perot (laser), 81–82
fiber-optic, for testing objectives,
240–241
Gires-Tournois, 88
Mach-Zender, to measure pupil function,
245
optical coherence tomography, (OCT),
504, 609
Twyman-Green, 239
Inter-fluorophore distance, measurement,
184. See also, Fluorescence
resonance energy transfer.
Interfocal crosstalk, 227–228.
disk scanners, 227–228, 444, 449
time multiplexing as solution to,
553–554
Interlocks, laser safety, 118.
Intermediate optical systems, LSCMs,
chapter, 207–220.
Internal focusing elements, in objective,
157, 511.
International Commission of Non-Ionizing
Radiation Protection (ICNIRP), 117.
International Electrotechnical Commission
(IEC), 117.
International television standards, 589.
Internet sources. See Links.
lasers, 123, 124
Intrinsic noise, 21. See also, Poisson noise.
Inverse filter algorithm, 476, 477.
Ion-binding in Aequorin emits light, 737.
Ion concentrations, 346–347, 517, 528–530,
741.
chapter, 736–745
determination, 517, 528–530
957
Ion-concentration imaging, 736–738,
740–745. See also, Calcium imaging,
pH, etc.
calcium imaging, 736–737
concentration calibration, 742–745
indicator choice, 738
interpretation, 740–741
pH imaging, 739–745
water-immersion objectives, 737
Ion sensitive probes, optical, 348, 737.
table, 531–532
IR. See Infrared; Near infrared.
Irradiance, arc and halogen light sources,
130.
table comparing, 130
ISO standard, microscope dimensions, 156.
Iso-intensity surface, or arc sources, 304.
Iterative constrained algorithms, 475–476.
See also, Deconvolution; Nonlinear
constrained iterative deconvolution
algorithms.
Iterative constrained Tikhonov-Miller
algorithm (ICTM), 497.
J
Jablonski energy diagrams, 516, 517, 697,
792.
Jansson-van Cittert algorithm, 476, 496.
Jitter, defined, for scanners, 54.
JND. See Just noticeable difference.
Joint Photographic Experts Group. See
JPEG.
JPEG (Joint Photographic Experts Group),
581–584.
Just noticeable difference (JND), ocular
response, 72–73.
K
Kaede, photoactivatable fluorescent protein,
emission change after photodamage,
187, 383, 385.
example image, 187
Kalman averaging, 21, 39, 53, 304, 306,
627, 638, 655, 750, 754, 781.
comparison with deconvolution, in
reducing
intensity, 39
Kepler, Johannes, 788.
Kerr cell, 516.
mode-locking (KLM), 111, 133
of titanium:sapphire lasing rod, 113
Kerr effect, defined, 111, 179.
self-focusing of pulsed laser light, 111
Kindling proteins, 574, 760.
Kinetics, 691, 694–698, 741–742, 774, 796,
810–812, 816–817.
bleaching, 691, 694–698
and endpoint data analysis, 816–817
fluorescence, 262–263, 348, 383, 385,
571, 578, 741–742. See also, FLIM
FRET, 796
high content screening, 810–812,
816–817
958
Index
Kinetics (cont.)
ion concentration dyes, 741
and STED, 571, 578
Kino, Gordon, confocal design, 6.
KLM. See Kerr lens mode-locking.
Köhler illumination, 34, 127–128, 131, 229,
251, 627, 648–649.
coherence of light, 131
in disk scanner, 229
field diaphragm, 35, 127–129, 139, 461,
627, 645, 648–649
to limit non-uniformity of illumination,
127–128
to measure photon efficiency, 34
Krypton laser, 102, 119, 346, 355.
comparison with argon-ion laser, 102
wavelength, 102
Krypton/argon (Kr/Ar) laser, 90, 92, 93,
102, 108, 119, 203–204, 343, 375,
748, 798, 811.
stabilization, 88
KTP. See Potassium titanium oxide
phosphate.
L
Labeled structures, plants, 757, 761, 775.
bulk labeling, living embryos, 761
cell walls, 775
selective labeling, 757
Label-free microscopy, noise, 114.
Lamp housing, 134.
Lamprey.
labeled axons, 235, 236
larva, optical projection tomography
image, 612
Landmark-based registration synthesis
method, 328–329.
Lanthanide chelates, 345–346.
Large mode area photonic crystal fiber
(LMAPCF), 110.
Larmor frequency, MRM imaging,
618–622.
Laser(s), 7–9, 44, 80–83, 88, 90, 94,
112–114, 119–120, 131, 540–543,
599–600. See also, Fiber lasers;
Mode-locked lasers; Multi-photon
ultrafast lasers; Up-conversion fiber
lasers; Ultrafast lasers.
Alexandrite, 109
amplifier rods, 116
attenuation of, 85, 87–88, 354, 415, 904
axial or longitudinal modes, 83
basic operation, 81–83, 116
CARS microscopy requirements, 599–600
chapter, 80–125, table, 119–120
coherence, spatial and temporal, 83–84
colliding-pulse mode-locked (CPM), 540
for confocal, 7, 9–10, 77–78, 280,
535–545
continuous-wave, 90–110
control of power, 543
Cr:Forsterite, 109, 114, 415, 541,
706–709, 712–714
excitation wavelength choice, 540–542.
See also, Acousto-optical devices,
filters
femtosecond pulsed laser, 44. See also,
Ultrafast lasers
fiber-based lasers, 109–111, 113–118
table, 94
ultrafast, 113–114
up-conversion fiber lasers, 109–110
fiber light delivery, 107,See also, Fiberoptics
GaAs, 107, 111
gas, 90, 91–10. See also, lasers by gas.
alkali-vapor, 103
Ar-ion, 90, 101–102
Kr-ion, 102
HeNe, 102–103
HeCd, 103
heat removal, 84
hybrid mode-locked dye laser, 540–541
important properties for confocal, 80
light delivery, 87–89
fiber-optic, 106
mirrors, 88
longitudinal modes, 82–83
maintenance, 115–116
active media replacement, 115
cooling components, 116–117
optical resonator, 116
metal vapor, 112
microscopical uses
nonlinear: 2- 3-photon, 90
Raman and CARS, 90
TIRF, 90
tweezers, 89. See Laser trapping
multi-photon. See Multi-photon
microscopy
Nd:glass, 706–708
Nd:YAG, lasers, 88–89, 91, 95, 97, 103,
107–109, 111, 113–115, 117, 218,
245, 514, 680, 798
Nd:YLF, lasers, 89, 98, 100, 103, 109,
112–114, 750, 760–761
Nd:YVO4, lasers, 89, 95, 100, 103,
107–109, 111, 113–114, 541
NO SMOKING, 116
performance tables, 91–101
phase randomization, 8, 13, 131–132, 143
pointing error, 87
active cavity stabilization, 87
polarization, 83, 88–89
power control, 543
pulse broadening/compensation, 88,
901–904
pulsed, 110–115. See also, Titaniumsapphire, Cr:Forsterite,
Nd:glass,YAG/YLF/YVO4, etc.
cavity dumped, 111
Kerr lens mode-locked, 111
modulated diode lasers, 112
pulse-length measurement, 115,
901–903
purpose, 110
saturable Bragg reflector, 111
ultrafast, DPSS lasers, 112
ultrafast, fiber lasers, 113
white-light continuum lasers, 113
why are they useful?, 110
pumping power requirements, 82
safety, 117–118, 839, 900. See also,
Safety
goggles, 118
screens and curtains,118, 904
solid state, 103. See also, Solid-state
lasers
semi-conductor, 105–107
thin-disk lasers, 109
spectrum of light, 44
stabilization, 85–87
active, 87
titanium:sapphire laser, 82, 84–86, 88–91,
94, 100–103, 105, 107, 109,
111–112, 114, 123–124, 165, 346,
358, 415, 423–424, 459, 541, 550,
551, 645–647, 688, 706–708, 713,
727, 750, 756, 759
4Pi, 563–564, 567
brain slices, 731
CARS, 599
compared to other fast lasers, 82–83,
85, 110, 112–113
embryos, 731, 750, 756, 759, 764
maintenance, 116
and OPO, 114–115
plants, 415, 423–424, 706–708,
713–714, 717, 781–783
popular models, specs, table, 120
STED, 575
transverse modes, 82–83, 85, 110
tweezers, 89. See Laser trapping
types, 90
ultrafast fiber, 113–114, See also,
Ultrafast lasers
wavelength expansion by sum-anddifference mixing, 114
optical parametric oscillators, 114–115
second/third harmonic generation, 114
white light continuum lasers, 88, 109, 113
continuum, 88, 109
He:Cd, 113.
Laser cavity stabilization, active, 87.
Laser cutters, 686–687.
integration, 218–219
Laser illumination, conditions for, 8.
Laser lines, using acousto-optical tunable
filters, 56.
Laser media, maintenance, 115–116.
Laser printers, 593.
Laser rods, maintenance, 116.
Laser Safety Officer, 117.
Laser sources, 9, 80–125. See also, Lasers.
Laser speckle, 84, 90, 130–132, 448.
removing, 84. See also, Scramblers
source, 130
Laser trapping, 80, 89, 110, 218–219, 383,
385, 539, 646, 680.
Index
Laser tubes, operational lifetime, 102, 115.
components likely to fail, 115
Laser tweezers. See Laser trapping.
LaserPix, 282.
Laser flying-spot microscope, 7.
Lasersharp, confocal microscopes, 282, 284,
285, 288, 292, 296, 302–306.
LaserVox, 281–282.
Lateral chromatic aberration (LCA), 14,
155–156, 239, 242–243, 287, 640,
657–658.
correction in conventional optics, 155
measured, 657–658
Lateral coherence, 8, 84, 267.
Lateral resolution, 1–4, 9, 11–13, 28, 207,
209, 222, 225, 230, 238, 270, 320,
409, 453, 511, 513, 542, 552, 554,
563, 568, 651, 654–656, 747. See
also, Resolution.
4Pi, 568
CARS, 596–597, 599
confocal endoscopy, 511, 513
confocal optics, improvement, 9, 651,
654–656
of display, 292
light microscopy, 1–3
optical coherence tomography, 609–610
with pinhole and slit disks, 225
and spherical aberration, 409
SPIM, 613, 674
STED, 573–575, 578
table, 209, 409
Laterally-modulated excitation microscopy,
see Stuctured-illumination.
LCA. See Lateral chromatic aberration.
LCD. See Liquid crystal display.
LCOS. See Liquid-crystal-on-silicon.
LCS (Leica Microsystems AG), 282, 312,
910.
Lecithin myelin figures, CARS image, 204.
LED. See Light-emitting diode.
Leica, confocal manufacturer, 51–53, 56–57,
160, 218, 797, 910.
acousto-optical beam-splitter, 160, 218
objective lens transmission, 160
RS Scanner, 52–53
spectral confocal, TCS SP2, 51, 56–57,
910
tube length conventions, 157, 239
Leica Microsystems AG, 282, 910.
Leica TCS 4Pi, 119–120, 565–568.
4Pi microscopy type C, 565–568
imaging of living cells, 568
lateral scanning, 567, 910
mitochondrial network image, 568
optical transfer function (OTF), 567
sketch, 566, 910
thermal fluctuations minimized, 567
Lempel-Ziv-Welch (LZW), 580–582, 584.
Lens aberrations, 13–15. See also,
Aberrations.
Lens focal length, change, with wavelength,
152.
Leonardo da Vinci, early optical studies,
788–790.
Leukocytes, 347, 387, 520, 815, 854.
automatic analysis, 815
multi-photon, phase-based FLIM, 521
Lifetime. See Fluorescence lifetime imaging
microscopy.
Ligand-binding modules, 256, 348, 741,
846.
Light detection, general, 28–33, 251–264.
See also, Detectors; specific
detectors: CCDs, PMTs, etc.
assessment of devices, 260–262
charge-coupled device (CCD), 254
comparison, table, 233, 255–256, 647
conversion techniques, 259–260
direct effects, 252
future developments, 262–264
history, 262–264
image dissector, 254–255
microchannel plate, 232–233, 255, 262
gated, 519, 523–524, 527, 532
MCP-CCD, 262
noise internal to, 256–259
internal detection, 256
noise currents table, 256
photoemissive devices, 256–257
photon flux, 257–258
pixel value representation, 258–259
photoconductivity, 252, 253
photoemissive, 254
photon interactions, 252–256
work functions, table, 252–253
photovoltaic effect, 252–253
point detectors, 260–261. See also, PMT
quantal nature of light, 251–252
thermal effects, 252
vacuum avalanche photodiode, 254, 255
Light dose, related to pixel/raster size, 64.
Light, effects, on plant cells, 770. See also,
Bleaching, Phototoxicity.
Light-emitting diode (LED), 34, 54,
132–133, 135–139, 143, 237.
aligning, 135
control by current-stabilized supply,
138–139
definition, 105
to detect galvanometer rotor position, 54
excitation wavelength for fluorophores,
136
expected cost reduction, 237
fluorescence image, 142
galvanometer position feedback, 53
lifespan, 137
to measure photon efficiency, 34
microscope illumination, 131–139, 141,
143
organic, projected development, 143
radiance, 138
spectra, 133
stability, 136
temperature effects, 137
wavelength vs. current change, 137
959
Light flux, light-emitting diode temperature,
133.
Light intensity, 71, 163.
Light microscopy history, 1–4.
Light paths. See also, Commercial confocal
light microscopes.
separating excitation/emission, 44–45
Light piping by specimen vs. depth, 182.
Light-sheet illumination, 672–673.
Light sheet microscopy, 613.
chapter, 672–679
optical setup for, 613
white-light continuum lasers, 113
Light sources, widefield, 132–139, 143. See
also, Chapters 5 and 6, Arc lamps,
LEDs, Lasers; Nonlaser light
sources; Filaments; Halogen.
commercial sources, 143
solar, 126–127, 131, 135
stand-alone, 143
table, comparative performance, 140
types, 132–139
Light transmission, 11, 139, 160–161,
223–229.
cummulative loss along optical path, 139
of Nipkow disk system, 11, 223–229
specifications for objectives, table,
160–161
Lighting models, 3D image display,
306–312.
absorption, 309–312
advanced reflection models, 309
artificial lighting, 309–312
Gourard shading, 308
gradient reflection models for voxel
objects, 309
Phong shading, 308–309
Phong/Blinn models, 308
simulated fluorescence process, 310
surface shading, 310
transparency, 280, 284, 287, 300, 304,
309, 311–312
Lilium longiflorum, image, 783.
Limitations, confocal microscopy, chapter,
20–42.
fundamental, 20–42
table, 41, 647
typical problem, 21, 24
Linear galvanometers, 54.
Linear longitudinal chromatic dispersion
(LLCD), stereoscopic confocal
image, 154.
Linear unmixing. See Spectral unmixing.
Line-scanning confocal microscope, 50, 51,
231–232, 237, 784, 908, 916.
Linearity, 72, 490.
deconvolution for image enhancement,
490
display advantages and disadvantages, 72
Links (Internet addresses).
2 photon excitation spectra, 546, 727,
729, 782
brain slices, 727
960
Index
Links (Internet addresses) (cont.)
CCDs, 76, 234, 927, 931
components, 58
confocal Listserve, 390, 901
deconvolution, 495
dyes, 221, 343–344, 782
fluorescent beads, 653
FRET technique, 185, 803
high-content screening systems, 811
image management, 865
lasers, 104, 115, 120, 123–125
live-cell chambers, 388–389, 870
movies related to book, 235, 392
muscles, 237
non-laser light sources, 138, 143
plants, 769
safety, 900
software, 282, 376, 594, 734, 762, 764,
776, 777, 820, 824, 827, 831–833,
844, 845, 864–862, 865–867, 869
SPIM, 672
Lipid dyes, 236, 355, 359–360, 389, 556,
755, 760–761.
Lipid receptors, 790.
Liquid crystal-on-silicon (LCOS), 266.
Liquid crystal display (LCD), 39, 67, 73,
291, 293, 589–590.
digital projectors, 590
filters, 928
non-linearities, 73
shutters, 299, 929
supertwisted nematic (STN), 589
thin-film transistor (TFT), 589
Liquid crystal technology/dynamic
polarization microscopy, 188. See
also, Pol-scope.
Lissajous pattern, circular scanning. 554.
“tornado” mode, SIM scanner, 52
List servers, 125.
Lithium triborate (LBO), as non-linear
crystal for multiplying infrared
output, 109, 115.
Living cells, 80, 90, 114, 136, 145–161,
167, 219, 221–222, 381–399,
429–439, 480, 564–566, 568,
746–766, 770, 772–773, 788–806,
811, 813. See also, Brain slices,
Plants cell imaging, and by
cell/organism name.
2-photon, penetration, 749–751
2D plus time, 753–754, 762–764
3D projection, 763
4D data, 746–747, 764
4Pi microscopy, 564–565, 568
acquisition speed, 222, 753–754
algorithms, 763–764
assays, 811
beauty and functionality, 790
bleaching of, 797. See Bleaching;
Photodamage
cell-chamber, 11, 22, 191, 219, 370–371,
386–387, 394, 429–430, 564,
610–611
for 4Pi confocal, 564
for biofilms, 870–873, 875, 877, 880,
885
for brain slices, 394, 723, 727, 729
for epithelial cells, 370–371, 377, 386
finder chamber, 683
flow chamber, 870–873, 875, 877, 880,
885
for high-content screening, 810
for optical projection tomography,
610–611
perfusion, 394
for plant cells, 191, 429–430
simple, 22, 394
for SPIM, 613, 625, 673
table of required functions, 380
table of suppliers, 388–389
test chamber/dye, 654, 661
cell-cycle effects, 790
chromatin, 385, 390–392, 684, 693–695,
812
chromatin dynamics, 390–392
CNS tissue slice preparation, 393
confocal microscopy, 381–399, 746, 813
difficulties, 381
future directions, 398–399
considerations, 386–390
antioxidants, 390
experimental variables, table, 386
fluorescent probes, 387–389
maintenance of cells/tissues, 387
minimizing photodynamic damage,
136, 389
photon efficiency, 141–161, 389–390
in vitro preparations, see In Vitro
in vivo preparations, see In Vivo
contrast, 747
dyes, 748. See also, Dyes; Fluorophors
etc.
for rapid assessment, table, 360
embryos, imaging, 746–766. See also,
Living embryo imaging
external membranes, SHC image, 90
fluorescent staining, 393
microglia, 393
nuclei, living/dead cell, 393
fluorophore effects, 748
FRET imaging, chapter, 788–806
future, 221–222
handling data, 395–396
imaging techniques, 382–386, 394–395
low-dose imaging, 391–392
microglial cell behavior example,
392–398
no damage from SHG imaging, 114
online confocal community, 390
photon efficiency, 141–161, 389–390
phototoxicity, 390–391
assays for, 813
plant, 429–439. See also, Plant cell
imaging reflectance imaging, 167
second harmonic generation. See also,
SHG
of external membranes, 90
no damage, 114
test specimen for, 390
widefield, 646–647, 751–753
working distance, 5, 9, 129, 145, 154,
157, 198, 249, 511, 568, 598, 634,
673, 678, 727–728, 747, 774, 779,
781, 872
table, 158
Living embryo imaging, 749–751, 762–764.
aberrations caused by, 747
apparatus, 748
C. elegans, 746, 748
deconvolution helps confocal, 751–753
developmental changes, 746
Drosophila, 273, 675–676, 747–748,
751–752, 754, 756, 759, 804, 810
dyes, 748
introduction of, 755
embryo size vs. speed acquisition,
753–754
explants, 748–749
future developments, 766
fluorescent probe
four dimensional, 746–747, 749
cellular viability, 747–748
challenges, 762
dataset display strategies, 761–764
photodamage during, 746–748
high speed acquisition
disk-scanning confocal microscopy,
754
hardware, 754–755
light scattering, 747
optimal acquisition, parameters, 753–754
refractile specimens, 747
superficial optical sections, 748
thick specimens
effective strategies, 748–753, 755–761
inherent trade-offs, 747–748
selective plane illumination (SPIM),
751
“Test drives,” for living embryo imaging,
752.
widefield/deconvolution, 751–752
LLCD. See Longitudinal chromatic
dispersion.
LMA-PCF. See Large mode area photonic
crystal fiber.
Loading methods, fluorescent probe, 347,
358–360, 430, 732–734, 738, 739.
acetoxymethyl esters, 359, 360. See also,
Acetoxymethyl esters
ATP-gated cation channels, 359
ballistic microprojectile delivery, 360,
726, 803
direct permeability, 358–359
electroporation, 359–360, 795, 803
ion indicators, 738–739, 742
low level, 430
membrane permeant esters, 359–360
microinjection, 360, 361, 388, 739, 748,
755, 795, 803–804
Index
neurons, 722, 726, 730, 732–734
osmotic permeabilization, 359
peptide-mediated uptake, 359
plant cells, 769
stabilizing chemicals, 341–342, 362
transient permeabilization, 359
whole-cell patch pipet, 360
Local projections, display, 305–306, 307.
Location proteomics, 818.
Longitudinal chromatic aberration, 152–155.
Longitudinal coherence length, 7, 8, 84,
130, 131.
Longitudinal linear chromatic dispersion
(LLCD) objectives for 3D colorcoded BSL confocal, 154.
Long-pass filters, 43–44.
Low-voltage scanning electron microscope
(LVSEM), 846–847, 849–850, 852.
LSM. See Laser-scanning confocal
microscopes; Laser-scanning flyingspot microscope. 6–7
Lucoszs formulation, 273.
Luminescent nanocrystals, 343, 345.
Luminous intensity vs, color, dye molecule,
138.
LVSEM, 846–847, 849–850, 852.
LysoTracker Red DND-99, 359–360,
709–710.
rapid assessment table, 360
spectra, 710
LZW compression. See Lempel-Ziv-Welch.
M
Mach-Zehnder interferometry, 245.
Machine learning. See Automated
interpretation of subcellular patterns.
Macrography, 3D light scanning, 672.
Magnesium fluoride (MgF2).
for anti-reflection coating, 158
Magnetic disks, 586.
Magnetic resonance imaging (MRI), 618.
Magnetic resonance microscopy (MRM),
618–624.
amplitude modulation for RF carrier, 620
applications, 623–624
botanical imaging, 624
developmental biology, 624
histology, 623
phenotyping, 623
basic principles, 618–619
Fourier transform/image formation, 620
future development, 624
hardware configuration, 621–622
image contrast, 622–623
image formation, 619–621
Larmor frequency, 620
Schematic diagram, 618–619
strengths/limitations, 622
Magnification, 24, 35–41, 62, 131, 215, 443.
See also, Nyquist sampling; Oversampling; Undersampling.
calibrating, 653, 658
and CCD pixel-size, 62, 70
confocal, 52–53, 62–64
effect on pixel size, 24, 928
factor, 24, 28
and lateral chromatic aberration, 278
for line-scanner, 232
over-sampling, 68–70, 493, 509, 635, 729
high-content screening, 816
and pinhole size, 28
under-sampling, 68
zoom magnification, 11, 24, 37, 63–34,
66, 70, 79, 317, 389, 493, 627,
634–636, 731
Maintenance.
cell viability, 387
dye lasers, 114
lasers, 115–117, 124
remote logging of, 864
troubleshooting reference, 124
Maize (Zea mays), 167–168, 172, 179, 202,
417–424, 428, 438, 710–711,
713–714.
2-photon, time-lapse microspectroscopy,
423
abnormal vasculature, 437
anther, 420, 433
attenuation spectrum, leaf, 418
cross-sections, stem, 172, 707
emission spectrum, 710, 711, 713
fluorescence spectra, 422–424
leaf,
attenuation spectrum, 418
optical section, 172, 179
reflectance, 167
surface, 436
meristem, 420, 430–432, 707
multi-photon excited signals, 422–424
polarization microscopy, 707, 711
pollen grain, 202, 433–434
protoplast, 424
root, 432
second harmonic imaging, 707, 711
silica cells, 428, 437, 707
spectrum, 422, 423, 710
starch, 420, 435–436, 707, 711
stem
attenuation spectra, 417, 418, 713
optical sections, 419, 714
storage structures, 420, 435–436, 707,
711
Manufacturers. See also, Commercial
confocal light microscopes;
Appendix 2.
listing with web addresses, table,
104–105.
Mapping conventions, in image processing,
294–296, 300–304.
data values, 300–304
choosing data objects, 300–301
object segmentation, 301–302
projection rules, 302–304
scan conversion, 301–302
table, 300
visualization, 300
961
multi-dimensional image display,
294–296
G function, 294
image/space view, 296
orthoscopic view, 294
reducing geometric dimensions, 294
rotations, 294–296
visualization process, 294
MAR. See Mark/area ratio.
Marching cubes algorithm, 301–302, 304,
776.
Marconi, CAM-65 electron multiplier CCD
camera, 76. See also, EM-CCD.
Mark/area ratio (MAR), 279.
Marsilea quadrifolia, 416, 419.
attenuation spectra, 416
optical section, 419
Mass balancing, to reduce scanner vibration,
54.
Mass storage, 580–588, 593–594.
data compression for, 288–289, 292–293,
295, 319, 499, 580–585. See also,
Data compression
algorithms, 319, 580
archiving systems, 580
color images, 581
file formats, 580–588
removable storage media, 585–588. See
also, Removable storage media
random-access devices, 586–588
sequential devices, 585–586
solid state devices, 588
time required, table, 581
Materials, silicon, fused quartz, beryllium,
52.
Mathematical formulas, for confocal
microscope performance, table,
209.
Maximum intensity projection, 180,
284–285, 292, 294, 298, 302–304,
307, 313–314, 319, 325–326,
330–331, 585, 755, 763–764, 770,
774, 881, 884.
local, 305
Maximum likelihood estimation (MLE),
472–475, 495, 497–498, 669.
blind deconvolution, 472–475, 498, 784
effect on colocalization, table, 669
M-CARS. See Multiplex CARS
microspectroscopy.
MCP. See Microchannel plate, 232–233,
255, 262.
MCP-CCD, 262
Gated intensified, 519, 523–524, 527,
532
MCP-PMT. See Microchannel plate
photomultiplier.
MDCK cell, 372–374.
actin cytoskeleton, 374
Golgi apparatus, image, 374
morphologic changes, 374
stereo image, 373, 374
vertical sections, image, 372
962
Index
Measurements, 20, 33–36, 76, 139–141,
159.
achromat performance, 194
buffering of, ion measurement, 738, 740
field flatness, 26–28
geometric distortion, 653–654
laser pulse length, 109, 112, 115, 507,
537, 538, 902–903
light throughput, 139–141
limits on confocal intensity, accuracy, 20
photon efficiency, 33–36
pinhole, effective size, 34
intensity spread function histogram,
74–78
resolution, 241–245, 657, 658
shrinkage, specimen preparation,
371–373
spectral transmission of objective, 159
spherical aberration, 145, 407
surface height, using LLCD BSL
confocal, 224
z-resolution, 194
Mechanical scanners, 51–54.
Melles Griot catalog, real lens, performance,
210.
Membrane permeant esters, 361, 358–359,
361, 726, 738–739, 744.
Membrane potentials, 179, 188, 204–205,
346, 353, 383, 517, 743, 811–813.
Memory stick, 588.
Mercury arc lamp, 37, 44, 132, 135–138.
fluorophores matching excitation,
135–136, 139
iso-intensity plots of discharges, 132
and pinhole size, 37
radiance, improvements, 137–138
wavelengths, 44
Mercury-halide arc source, 136, 138,
143–144.
spectrum, 144
Mercury-iodine (Hg-I) arc lamp, radiance,
138.
Mercury-xenon arc lamps, 136–138.
spectral lines, 136
Meristem, 168, 420, 430, 432, 770,
776–778, 782.
maize, 168, 432
Merit functions, confocal scanners, 217.
object-dependent, defined, 217
object-independent, defined, 217
Mesophyll cells, 169, 193, 195, 417–418,
423, 428, 430, 711–712, 714, 779.
A. thaliana, 193, 196
photodamage, 203
protoplasts, 196, 203, 424, 425–426,
430, 439
harmonic images, 711–712, 714
image, 424
spectra
attenuation, 416, 418
change with 1- vs. 2-photon, 421, 423
emission, 423
Metal-halide light source, 136, 143–144,
907, 908.
Metal vapor lasers, 112.
Metamorph, 281–282, 290, 311, 817.
Microchannel plate (MCP) image intensifier,
233, 255, 519, 532.
multiplicative noise, 233
photocathodes, 262
PMT, 255, 523, 532
Microchannel plate PMT (MCP-PMT), 255.
Micro-computerized tomography (MicroCT), 614–618.
contrast/dose, 614–615
dose vs. resolution, graph, 616
layout, 614
mouse images, 615–617
tumor-bearing, 617
operating principle, 614
Micro-CT. See Micro-computerized
tomography.
Microdissection.
with multi-photon IR light, 686–687
with nitrogen lasers, 112
Microelectrodes, for introducing indicator,
738.
Microglial cell behavior, 392–398.
Microinjection, 360–361, 388, 739, 748,
755, 795, 803–804.
of chromophores, 803–804
Microlens array, 12, 134, 135, 216, 225,
231, 235.
for 4Pi confocal, 563–565
for CCD, 237
for disk scanners, 6, 12, 216, 224, 226,
231, 458
for light-emitting diode source, 134–135
for multi-focal, multi-photon, (MMM),
537, 551–555, 558
principle, 135
in Yokogawa disk-scanning confocal, 12,
224–226, 231, 235
Microscopes, 217, 226. See also, particular
types.
attachment of confocal scanner, 217
specification comparisons, table, 226
Microscopy laboratory URLs, 125. See also,
Links.
Microspectroscopy, 421–425, 426, 516.
CARS, 601–602
fluorescence properties of plants, 421–425
lifetime, 516
of maize, 424
multi-photon setup, 424
Microspores, birefringence images, 189,
431–432.
Microsporogenesis, 431–432.
Microstructure fibers, 504.
Microsurgery, 112, 219, 686–687, 764–765.
Microtubule, 11, 68, 80, 188, 222, 292, 432,
582, 703, 714, 752–753, 759, 773,
790, 852. See also, Cytoskeleton.
birefringence, 714–715
Brownian motion of, 11
electron microscopy, 848, 850
fixation, 369, 372–375
fluorescence correlation spectroscopy, 383
GFP, 12. See also, Green fluorescent
protein
in mitosis, 759. See also, Mitotic
apparatus
polarization microscopy, 15, 173, 188,
420–421
photodamage of, 341, 850–851
stabilizing buffers, 852
STED, 576–577
stereo image, 752
TIRF, 180, 183
second harmonic generation, for tracking,
90
Microwave fixation, 377–378.
Microwire polarizer (Moxtec Inc.), 85.
Mie scattering, 162–163, 167, 417–418.
clearing with index-matched liquid, 167
comparison with Rayleigh scattering,
163
light attenuation in plant tissue, 417
by refractive structures, 162–163
MII. See Multi-photon intrapulse
interference, 88.
Mineral deposits, plant, 163–420, 436–437,
703.
Miniaturized fiber-optic confocal
microscope, 508–512.
bundle imagers for in vivo studies, 509
clinical endoscope, 514
objective lens system, 509
optical efficiency, 509
optical schema, 508
resolution, 509
rigid endoscope, 511
single fiber designs, 510
vibrating lens and fiber, 510–511
in vivo imaging in animals, 512
Minolta, CS-100 radiospectrometer, 139.
Minsky, Marvin, 2, 4–6, 11, 141, 216, 890.
Mirror coupling, pulse width and pulse
shape, 88.
Mirrors, 26, 48, 54, 63, 209–210, 214.
galvanometer, 54. See also,
Galvanometers
internal, testing reflectance losses, 26
laser-line, 48
performance, 54, 63.
scan angle and magnification, 63
size calculation for LSCM, 209
x-y scanning mirror orientations, 214
Mismatch, 893.
probe shape/pixel, 39, 466
caused by chromatic aberration, 243
refractive index, 377, 404–412, 411, 654,
658, 747, 863, 893
4Pi, 568
causing signal loss, 148–150, 408–409,
654
chapter, 404–412
corrections, 411–412
embryos, 747
film vs. CCD, 590
harmonic signal generation, 704–705
less, at long wavelength, 416
Index
measurement, 148–150, 655–656
of movie frame rate, 839
MRM, contrast agent vs. imaging time
mylar flakes, 198
resolution loss measured, 192–194
vector mismatch in CARS, 596–597,
600
z-distortion, 287
Mitotic apparatus, 15, 173, 373–374, 377,
386, 421, 431, 693, 749, 752, 799.
See also, Microtubules.
damage, 693
fixation, 373–374, 377
FRET, 765
marker, cyclin-B, 790
Pol-scope, 13, 188, 432, 468, 479–480.
deconvolved, 479
images, 15, 188, 479, 717
SHG imaging, 702, 718–719
in vitro fertilization, 188
Mitotracker stain, 142, 170, 353, 358, 360,
430–431, 692, 750.
living cells rapid assessment, table, 360
Mixing, sum or difference, to generate laser
wavelengths, 114.
MLE. See Maximum likelihood estimation.
MMM. See Multi-focal, multi-photon
microscopy.
MMM-4Pi microscopy, 556.
MO (magneto-optical) disks, 586.
Model-based object merging, 323–325.
Mode-locked lasers, 87, 101, 111–114, 118,
124, 358, 520, 646, 728–729, 749,
901–904.
active, pulsed laser class, 111
adjustment of, 901–904
for CARS, 599–600
colliding pulse, 112
fiber, ytterbium and neodymium,
113–114
fiber/diode, ultrafast, 113–114
FLIM, 520
interference with, by specimen, 171
Kerr lens, 111
modulator, fiber lasers, 111
multi-photon, 535–536, 540–541,
550–551, 563–564, 567, 646,
728–729, 749
passive, 111, 113–114
saturable Bragg reflector, 111
SHG, THG, 706–707
Mode-locked oscillators. See also, Modelocked lasers.
nanojoule pulse energies, 111
Moiré effects, 270–271, 755.
ambient fluorescent room lighting, 201
banding patterns, 755
disk-scanners and CCDs, 231, 754–755
structured-illumination methods, 268–271,
273
Molecular imaging, in vivo, 387, 618, 624,
790, 803–806.
FRET, 790, 803–806
micro-CT, 618
MRM, 624
Monitors, computer display, 588–589.
Monkey cells, 693, 803.
Monomeric red fluorescent protein (mRFP)
constructs, 756, 760, 798.
with CFP or GFP molecules, as FRET
pair, 798
Montage synthesis method, 281, 312, 318,
328–331, 748, 753, 851–852, 855,
858–859.
defined, 329–330
examples, 330–332, 780–781
scanning electron micrographs, 851–852,
855
TEM methods, 858–859
Moon, early phase measurements, 788, 789.
Morphological filters, 285, 300–301,
316–317, 320–322, 730–734, 817,
826.
high-content screening, 812, 819, 826
Morphometry, 145, 316, 319, 331, 726, 728.
group properties, 331
intensity/spectral measures, 331
interest points, 331
invariants, 331
location/pose, 331
shape measures, 331
size measures, 331
texture measures, 331
topological measures, 331
Mosaicing. See Montage synthesis method.
Mounting medium, 166, 198, 342, 370–371,
373–377, 404–413, 418, 454, 457,
473, 493–794, 499, 564, 631, 642,
652, 655, 696, 730, 774 See also,
Clearing agents.
brain slices, 730
chapter, 404–413
clearing solutions, 166, 417–418, 420,
439, 610, 624, 774–775
effect of glass bead, 199
plant specimens, 418, 431, 774
refractive index, tables, 377
selection, 198, 631
Mouse, 192, 376, 393, 608, 612, 615+, 723,
726.
confocal colonoscopy, 509, 512
embryo
optical projection tomography image,
612
SIM/EFIC image, 608
GFP transgenic, 726
hippocampus, 393
micro-CT image, 614–615, 617
femur, 616
tumor, 617
spectral unmixing image, 192, 382,
664–667
examples, 665–666
visual cortex brain slices image, 723
Movement contrast, 190.
Movie compression, 836–840.
Moving-coil actuators, galvanometer, 52.
Moxtec Inc., Microwire polarizer, 85.
963
MPA. See Multi-photon absorption.
MPE. See Multi-photon excitation.
MPEG display formats, 836–841.
MPEM. See Multi-photon microscopes.
MPLSM. See Multi-photon laser-scanning
microscopy.
MPM. See Multi-photon microscopy.
MQW. See Multiple quantum wells.
mRFP. See Monomeric red fluorescent
protein.
MRI. See Magnetic resonance imaging.
MRM. See Magnetic resonance microscopy.
Multi-channel experiments, 813.
filters and dispersive elements, 51
time-lapse fluorescence imaging, 382,
384
toxicity, 755
Multi-dimensional microscopy, display,
280–314. See also, Automated 3D
image analysis methods.
2D pixel display space, 291
efficient use, 292
animations, 292–293
artificial lighting, 306–308
CLSM images, 286–290
anisotropic sampling, 287
calibrating image data, 286
data type/computational precision,
288–289
dimensions available, 286–287
file formats for
calibration/interpretation, 288–289
image data, 286
image size available, 287
image space calibration, 287–288
image/view dimension parameters,
table, 288
processor performance, 289–290
storing image data, 286
voxel rendering speed, 290
color display space, 291–292
commercial systems, tables
available systems, 282–283
desirable features, 288–289
display options, 293
geometric transformations, 295
projection options, 300
realistic visualization techniques,
307
criteria for choosing visualization, 281
data values, definition, 222, 280
dimensions, 280, 323
degrees of freedom, optical image, 8–9
depth-weighting, 304, 306
exponential, 304
linear, 304
recursive, 304
display view, definition, 280
hidden-object removal, 304–305
local projections, 305–307
z-buffering, 304–305
highlighting previously defined structures,
284
image, definition, 280
964
Index
Multi-dimensional microscopy, display
(cont.)
image/view display options, table, 293
geometric transformations, table, 295
intensity calibration, 304
iso-intensity surface, 304
laser-scanning microscopy, 280
lighting models, 306–312
absorption, 309–312
advanced reflection models, 309
artificial lighting, 309–312
Gourard shading, 308–309
gradient reflection models/voxel
objects, 309
Phong shading, 308–309
Phong/Blinn models, 308
simulated fluorescence process, 310
surface shading, 310
transparency, 309–312
living cells of rodent brain, 392–398
mapping data values, 300–304
choosing data objects, 300–301
object segmentation, 302
projection rules, 302–304
scan conversion, 301–302
segmenting data objects, 301
visualization model, 300
mapping into display space, 294–296
G function, 294
image/space view, 296
orthoscopic view, 294
reducing geometric dimensions, 294
rotations, 294–296
visualization process, 294
measurement capabilities See also,
Chapter 15
reconstructed views, 312–313
results, 284–285
objective vs. subjective visualization, 281
prefiltering, 281
principle uses, 281–285
projection/compositing rules, 302–304
alpha blend, 302, 304
average intensity, 302
first or front intensity, 302
Kalman average, 304
maximum intensity, 302
pseudo color, 173–175, 190, 291
purpose, 281–285, 293–295
realism added to view, 306–308
techniques for, table, 307
reconstructed view generation, 290–312
5D image display space, 291–294. See
also, 5D image display space
choosing image view, 291–294
subregion loading, 290–291
reconstruction, definition, 280
reflection models, 306–308
rendering, definition for, 280
software packages, table, 282–283
stereoscopic display, 293, 296–299
color space partitioning, 297
interlaced fields of frame, 297
pixel-shift/rotation stereo, 297
stereo images example, 298
synchronizing display, 297
true color, 291
unknown structure identification, 281–284
viewing data from, 283
visualization parameters, table, 285
z-coordinate rules, 304
z-information retained by, 296–300
non-orthoscopic views, 299
stereoscopic views, 296–299
temporal coding, 299–300
z-depth, 299–300
Multi-fluorescence, systems for utilizing,
217+.
Multi-focal, multi-photon microscopy
(MMM), 221, 276, 550–559, 797.
4Pi-MMM, 563–564
basics, 565
scheme, 563
alternative realizations, 554–555
background, 550
beam subdivision approaches, table, 558
current developments, 558–559
experimental realization, 551–555
FRET, 797
imaging applications, 556
boar sperm cells, 557
Convallaria, 556
FRET, 556
hippocampal brain slices, 557
pollen grains, 556
Prionium, 556
interfocal crosstalk, 553–554, 556
time-multiplexing, 553–555
limitations, 556–558
localization, 538
Lissajous pattern of scanning foci, 554
“tornado” mode, SIM scanner, 52
Nipkow-type microlens array, 551–552
optimum degree of parallelization,
550–551
resolution, 552–553
schematic diagram, 552
time multiplexing, 553–554
variants, 555–556
FLIM, 555–556
MMM-4Pi, 556
SHG, 556
space multiplexing, 555
Multi-length fiber scrambler, 8. See also,
Scramblers, light.
Multi-photon absorption (MPA), 535.
Multi-photon excitation (MPE), 356–358,
535–545, 894. See also, Multi-focal
multi-photon microscopy.
absorption, 705–707
advantages/disadvantages, 644–647,
749–751
autofluorescence, plants, 424, 427
background from SHG/THG, 361,
708–709, 728
backscattered light imaging, 429
bleaching, 218, 338, 539–540, 680–689,
692–693, 905. See also, Bleaching;
Chapter 38
caged compounds, 187, 383, 543–544,
692, 729, 912
cell viability during imaging, 544–545
chromophores for, 543–544
detection, 538
duty cycle, 644
excitation localization, 538
excitation spectra, 125
FLIM, 576
fluorophores for, 543–544
FRET, 797
heating, 539–540
history, 535
image formation, 535–540
instrumentation, 540–543, 900–905. See
also, lasers for. See also, Ultrafast
lasers
Alexandrite, 109
Cr:Forsterite, 109, 114, 415, 541,
706–709, 712–714
Nd:glass, 706–708
Nd:YAG, 88–89, 107–109, 514, 680,
798
Nd:YLF, 89, 112–114, 750, 760–761
Nd:YVO4, 89, 95, 107–109, 113–114,
541
Ti:Sapph. See Laser, titanium-sapphire
laser
multi-focal, multi-photon microscopy
alignment, 900–901
beam delivery requirements, 541
control of laser power, 543
CPM laser, 540
descanned detection, 166, 208, 212,
428, 537, 540–542
excitation wavelengths, 541
focal plane array detection, 542
hybrid mode-locked dye laser, 540–541
lasers/excitation wavelength choice,
540–542
non-descanned detection, 185, 201,
218, 381, 447, 456, 507, 542, 552,
559, 643, 646, 727, 750, 779, 904,
909, 910
non-mechanical scanning, 543
optical aberrations, 542
power requirements, 541, 903, 904
pulse spreading due to GDD, 547, 538,
543
resonant scanning, 543
whole-area and external detection,
541–542
optical pulse length, 537–538
group delay dispersion, 537–538, 543
group velocity dispersion, 88, 111, 210,
537, 606, 903
measurement, 115, 901–903
penetration, 749–750
photodamage, 539–540, 680–688,
692–693
Index
physical principles, 535–540
refractive index mismatch, 404–413
resolution, 539
SHG and THG background, 361,
708–709, 728
two-photon absorption cross-sections, 125
(URL) 543–544
wavelengths, 538–539
Multi-photon intrapulse interference (MII),
88.
Multi-photon microscopy (MPM), 10–11,
56, 172–177, 210, 535–545, 681,
682, 685–688, 746–766, 894,
900–905.
advantages/disadvantages, 644–647,
749–751
alignment, 901–902
autofluorescence, 425–427, 545
SHG, THG, 361, 708–709, 728
calcium imaging, 545
cell damage during, 544–545, 682, 685
1-photon vs. 2-photon excitation, 681
absorption spectra of cellular absorbers,
681
intracellular chromosome dissection,
688
mitochondria, 686
nanosurgery, human chromosomes,
686–687
by optical breakdown, 198, 680, 682,
685, 687, 703, 705
photochemical, 682–685
photothermal, 539, 545, 681, 685,
904
reproduction affected by ultrashort NIR
pulses, 686
ultrastructural modifications, 685–686
cell viability, 544–545
compared with other 3D methods,
644–647, 748–751
deconvolution, 495–498
developmental biology, 545, 746–754,
757, 759–760, 764
dispersion as problem, 56. See also,
GVD; GDD
fluorescence, contrast, 172–177
for living embryo imaging, chapter,
746–766
need for efficient illumination light path,
210
optical layout, 540
photobleaching, 545, 680–688, 692–693
practical operation, 900–905
protein damage/interactions, 765
resolution, 552
setup/operation, 540, 900–905
schematic diagram, 540, 901–902
in vivo (intact animal) imaging, 545
ultrafast lasers, 88, 90, 109. See also,
Ultrafast lasers
Alexandrite, 109
Cr:Forsterite, 109, 114, 415, 541,
706–709, 712–714
Nd:glass, 706–708
Nd:YAG, 88–89, 107–109, 514, 680,
798
Nd:YLF, 89, 112–114, 750, 760–761
Nd:YVO4, 89, 95, 107–109, 113–114,
541
Ti:Sapph. See Laser, titanium-sapphire
laser
uncaging, 545
Multi-photon-based photo-ablation, 764.
Multi-slit design, for disk-scanning
confocal, 229.
Multi-view deconvolution, 330, 675–677.
Multiple quantum wells (MQW), diode
injection lasers, 106.
Multiplex CARS microspectroscopy (MCARS), 601, 602.
Multiplicative noise, 28–33, 51, 77–78, 224,
234, 256–258, 262, 275, 443, 460,
633, 661, 667.
of EM-CCD, 30–31, 77–78, 264, 256,
262
losses in effective QE from, 33, 234,
443
from PMT, 29, 51, 77–78, 233, 256–258,
460, 633, 661, 667
and quantum efficiency, 33, 234, 443
photon counting, 32–33, 78
pulse pile-up, 32–33, 35, 78, 521, 523,
526–527
table, 256
why it is usually unnoticed in LSCM,
633, 661
Muscle, 737, 739–742.
fatigue, 739–740
N
NA. See Numerical aperture.
Nanobioscopy, protein/protein interactions,
795–798.
acceptor bleach, 797–798
donor fluorescence, 796–797
FRET measurement, 795
sensitized acceptor emission, 795–796
Nanoscale resolution with focused light,
571–578. See also, Stimulated
emission depletion (STED)
microscopy.
breaking the diffraction barrier, 571–573
different approaches, 573–574
ground state depletion (GSD), 573
STED, 573–574
outlook, 577
RESOLFT concept, 571–573
resolution, new limiting equation, 571
measured, 578
stimulated emission depletion (STED),
573–578
axial resolution increase, 576
compared to confocal microscopy, 576
dyes, suitable, table, 575
OTF comparison, 578
PSF comparison, 578
965
Nanosurgery, 219.
with multi-photon systems, 90
NCI60 CMA, standard encapsulation, 816.
NCPM. See Non-critical phase matching.
ND. See Neutral-density filters.
Near infrared (NIR) lasers, 10, 90, 106. See
also, Lasers: titanium-sapphire; Nd:;
Cr:Forsterite.
Near infrared (NIR), 10, 90, 106.
diode injection lasers, 106
for laser tweezers, 90
objective lenses designed, 174
Nearest-neighbor deconvolution algorithm,
476.
image enhancement, 495–496
Negative contrast, for fluorescence
microscopy, 173–174.
Negative feedback, to correct mirror motion,
53.
Neodymium glass laser, 706–708.
Neodymium-yttrium aluminum garnet
(Nd:YAG) lasers, 88–89, 91, 95, 97,
103, 107–109, 111, 113–115, 117,
245, 514, 680, 798.
infrared range, 108
pumping non-linear crystal/green light,
114–115
Neodymium-yttrium lithium fluoride
(Nd:YLF) laser, 89, 98, 100, 103,
109, 112–114, 750, 760–761.
Neodymium-yttrium orthovanadate
(Nd:YVO4) laser, 89, 95, 100, 103,
107–109, 111, 113–114, 541.
kits utilizing, 113
Nerve cells, images.
Alexa stained, 330
backscattered light images, 167
eye, optic nerve, 481
Golghi-stained, 298
Lucifer-yellow, 314
microglia, 396–398
rat-brain neurons, 398
transmitted light, 475
Neutral-density filters (ND), 43, 76, 126.
in fixed-pattern noise measurements,
76
to reduce source brightness, 43, 126
NFP. See Nominal focal position.
Nikon, confocal manufacturer, 13, 15,
119–120, 161, 199, 201, 507,
638–640, 657, 750, 910.
C1 confocal microscope, 119–120, 507
C1si spectral confocal microscope, 908,
910
CF objectives, 154–156, 217, 669, 779
confocal x-z, BSL image, 22
Plan Apo objective, 13, 15, 638
resolution, measured, 16, 638–640,
657
water-immersion lenses, 15
high-content screening, 810
tube length conventions, 157, 239
Nile Red, dye, 435, 528, 575, 774, 782
966
Index
Nipkow disk scanning, 2, 5–6, 11, 12, 41,
215, 223, 231, 276, 551, 754,
783–784, 810, 894. See also,
Yokogawa; Disk-scanning confocal
microscopy.
commercial systems, 907, 913, 915
compared to single-beam scanning, 458
for high-content screening, 810
micro-lens system, 6, 12, 216, 224–226,
231, 234, 237, 551–552
multi-photon, 537, 551–558, 563–565.
See also, Multi-focal, multi-photon
microscopy rotation, 754
for single-sided confocal, 6, 141, 223,
229
source brightness, 141
speed of image acquisition, 11, 220,
222–226, 227, 231
for tandem-scanning, 141, 215
visualization, of cells, 458, 667, 754,
784
Nipkow, Paul, 5–6, 109
NIR. See Near infrared.
Nitrogen lasers, 112.
nanosurgery using, 219
NLO. See Non-linear optical effects.
NMR. See Nuclear magnetic resonance.
Noise, 21, 28, 74–77, 83, 87, 114, 190, 232,
256–259, 442–444, 495. See also,
Signal-to-noise ratio; Poisson noise;
Quantum noise.
background, 443–444
of CCD detectors, 30–31, 77–78,
232–233, 256, 262
equations, 256
table, 256
vs. photomultiplier tube detectors, 74,
77
CIC, clock-induced charge, EM-CCDs,
234, 926
in counting quantum-mechanical events,
21
deconvolution reduced noise, 39–40, 114,
392, 495, 498, 667, 783, 835–836
detector, 28
fixed-pattern, 74, 76, 278, 924, 927, 931
in fluorescence microscopy, defining,
74–75
in lasers, sources, 85–86
reducing, 87
limits grey levels, 443
measurement, 74–75
multiplicative, 28–33, 51, 77–78, 224,
234, 256–258, 262, 275, 443, 460,
633, 661, 667
in photon detectors, 256–259
noise currents table, 256
photo flux, 257–258
photoemissive devices, 256–257
pixel value represented, 258–259
Poisson. See Poisson noise
polarization, in laser systems, 83
read, and readout speed, 77
shot, 442–443. See also, Poisson noise
single-pixel, 65, 67, 190, 635, 832,
835–836
deconvolving, to reduce, 39–40, 392,
498, 667, 784, 835–836
reducing, 39, 40, 190, 41, 65, 392, 498
sources of, 442–444
wavelet transform to reduce, 733–734,
819–820
Nomarski DIC contrast, 2, 368, 746, 892.
See also, Differential interference
contrast.
Nominal focal position (NFP), 405, 408,
409.
calculations for glycerol, 409
calculations for water, 409
z-responses, diagram, 408
Non-confocal microscopy vs. confocal, 746.
high content screening, table, 811
Non-critical phase matching (NCPM),
114–115.
Non-descanned detection, for MPM, 185,
201, 218, 381, 447, 456, 507, 542,
552, 559, 643, 646, 727, 750, 779,
904, 909, 910.
for CARS, 559
No-neighbor algorithm, 476–477, 496.
Non-laser light sources, chapter, 126–144.
arc sources, 130, 132, 140
commercial systems, table, 143
comparative performance, table, 140
control, 138
for disk-scanning confocal, 141
filament sources, 135–136
LEDs, 132–133, 135, 138–139, 143
light scramblers, 131–132
measured performance, 139–141
results, 142
solar, 126–127, 131, 135
stability, 136–137
Nonlinear constrained iterative
deconvolution, 68, 458, 475–476,
496–497, 499, 520, 568.
Nonlinear conversion, tunable laser, 114.
Nonlinear crystals, frequency multiplying,
109.
Nonlinear optical (NLO) effects, in
microscopy, 90, 114, 163, 165, 177,
179, 188, 190, 195, 416–417,
426–427, 430, 442, 504, 535, 507,
703–720, 728, 741, 751. See also,
Multiphoton/microscopy; Harmonic
signals; SHG, THG.
absorption, 188, 415–418, 426–427, 430,
705
bleaching, 536, 550, 558, 645, 680–685,
693, 697, 707, 729. See also,
Bleaching; Photodamage
CARS, 595–598, 600
DIC, 473–474. See also, Differential
interference contrast
fluorescence, 172, 179
focus shift with spherical aberration, 409
harmonic generation, 704–705
emission, 710–711
energy state diagram, 705
multi-photon absorption/fluorescence,
705
second harmonic generation (SHG),
704–705
setup, 708–709
third harmonic generation (THG), 705
light sources/detectors, 706–708
light attenuation spectra in plants, 706
photodetector characteristics, 707
pulsed-laser, table, 706. See also,
Ultrafast lasers
in optical fiber, 504–508
optically active animal structures,
714–717
man-made collagen matrix, 717
signal-producing structures, table, 715
spindle apparatus, 717–718
zebrafish embryo, 716, 718
optically active plant structures, 710–714
Canna, 710
Commelina communis, 712
emission spectrum of maize, 710, 711
maize stem, 711, 714
potato, 712
rice leaf, 712, 715
polarization dependence of SHG, 717,
719
setup for, 708–710
spectra, 415, 417, 435
Euphorbia pulcherrima, 710
maize leaf, 710
Pyrus serotina, 711
STED microscopy, 571–579. See also,
STED microscopy.
structured illumination, 270, 276
Non-radiative dipole-dipole interactions,
790.
Non-specific staining, 27, 44, 74, 303, 345,
357–358, 442, 467, 472, 617, 660,
667–668, 760, 820, 878, 882. See
also, Background.
Non-tunable solid-state laser, 103.
Normal, free-running, pulsed laser, 111.
Northern Eclipse, software, 282.
Notch filter, to transmit laser line, 49.
Novalux Inc., Protera 488 laser system, 107.
NSDC. See Nipkow spinning-disk confocal.
Nuclear import analysis, 802.
Nuclear magnetic resonance (NMR), 618.
Numerical aperture (NA), 1, 4, 24, 28, 61,
126, 141, 145, 148, 168, 180, 195,
198, 239–250.
affects surface reflection contrast, 180
defined, 1
determining axial resolution, 4, 241–242,
657
determining lateral resolution, 1,
241–242, 656
diffraction orders accepted by, 61
effect on self-shadowing, 168, 198
Index
in fiber-based mini-confocal endoscopes,
509
image brightness, 126
matching to CCD pixel size, 62, 928
objective lenses with high, 145, 239–250
empty aperture, 248
with oil-immersion vs. water objective,
148
pinhole size as function, 28
and refractive index mismatch, 147–148.
See also, Spherical aberration in
tandem scanning confocal
microscopy, 141
vertical shadowing, 195
and zoom setting optimal, 24
Nyquist criterion, and digitization, 38–39,
64–68.
Nyquist digitizing, 65, 67.
Nyquist filtering, 70–79, 281.
Nyquist frequency, 64, 301. See also,
Shannon sampling frequency.
Nyquist, Harry, 64.
Nyquist noise, 256.
Nyquist reconstruction, limit output
bandwidth, 59, 66–67, 69, 70, 173,
235–236, 280–315, 458, 468–469,
474–475, 496–497, 563, 585, 603,
607, 610, 615, 635, 672, 675,
677–678, 690, 772, 730–731, 762,
77, 774–776, 778, 784, 883.
Nyquist sampling, 24, 37, 39, 40, 53, 60,
64–70, 73, 75–76, 78–79, 142, 146,
152, 205, 222, 258, 271, 273, 289,
386, 391, 448, 635–636.
blind spots, 38
for CCD camera, 70, 233, 273, 928
and deconvolution, 59, 65, 67–68,
222–223, 635
diagram, 60
optimal, results of deviating from, 24
practical confocal microscopy, 448,
635–636
reconstruction, see Nyquist
reconstruction.
relationship with Rayleigh-criterion and
PSF, 39, 60, 64, 66
signal-to-noise ratio, 67, 448
subpixel, resampling, 478–479
O
Object scanners, image quality, 216.
Objective lenses, 13, 15, 25–26, 34, 49, 145,
152, 156, 239–250, 652–660. See
also, Aberrations.
apodization, 250
axial chromatic registration, 287, 658
axial resolution measurement, 656–657
vs. pinhole size, 656
chromatic aberrations 14, 145, 152–156,
160, 177–178, 209, 242–243, 641,
659
apparatus in measuring, 243–244, 654,
659
axial shift, 243–245, 657–658
chromatic registration, 657–658
cleaning, 642
confocal performance, 145–161, 652–660
contrast transfer function (CTF), 16, 35,
37–39, 59–62, 656, 747
coverslip thickness, table, 654
dipping lenses, 161, 209, 411, 429, 568,
613, 727, 737, 870, 872
dry, high-NA, aberrations, 15
field illumination, 34–35, 127–128, 139,
461, 627, 658
flatness of field, 145, 151–152, 154, 418,
457, 639, 659
Focal CheckTM beads, 657
high-NA planapochromat, 13, 145,
239–250
infinity correction, 155–157, 166, 239,
405
advantages, 49
lateral chromatic registration, 657–658
lateral resolution. See CTF
light, vector nature, 267
mounting media. See Mounting media
photon efficiency losses, 25–26
plan objectives, table, 152
point spread function of high NA,
239–250
measuring, 240–242, 455, 462, 471,
656
polarization effects, 249–250
pupil function, measured, 245–248
3D point spread function restored,
247–248
empty aperture, 248
Mach-Zehnder interferometry, 245
phase-shifting interferometry, 245
Zernike polynomial fit, 245–247
table, 247
resolution test slide, 169, 656
spherical aberration. See Spherical
aberration
correction, 654–655
sub-resolution beads, 181–182, 196, 454,
477, 493, 499, 527, 652–656, 784,
900, 904, 930
images, 656
table of suppliers, 653
temperature variations, 248–249
transmission, optical, 154, 158, 159–161.
See Transmission, objective
table of objective lenses, 159–161
water-immersion, 145, 149–150
dipping objectives, 161, 209, 411, 429,
568, 613, 727, 737, 870, 872
use and limitations, 15
working distance, 5, 9, 129, 145, 154,
157, 198, 249, 511, 568, 598, 643,
673, 678, 727–728, 747, 774, 779,
781, 872
x-y and z resolution using beads, 656
OCT. See Optical coherence tomography.
OLED. See Organic light-emitting diodes.
967
Olympus, confocal manufacturer, 52–53, 54,
119–120, 161, 184, 187, 204, 229,
230, 234–236, 419, 421, 427, 557,
708–709, 727–730, 797, 908,
912.
Fluoview-1000, 119–120, 184, 187, 204,
908, 912
DSU disk-scanning confocal microscope,
229–230, 234–235, 908, 913
FRAP system, 210
FRET, 797
high content screening, 811
objectives, 557, 727–730
stick, in vivo objectives, 806
TIRF objectives, 183
transmission, table, 159, 161
SIM scanner, 52–54
tube-length conventions, 157, 239
On-axis reflections, artifact, 171.
Onion epithelium (Allium cepa), 390.
Online confocal community, Listserv,
390.
OPA. See Optical parametric amplifiers.
OpenLab, 282.
Operational lifetime, of laser tubes, 102.
OPFOS, Orthogonal-plane fluorescence
sectioning, 672–673.
OPO. See Optical parametric oscillators.
OPT. See Optical projection tomography.
Optical aberrations, 109, 542. See also,
Aberrations.
thin-disk laser optics, 109
Optical layout of confocal microscopes,
212–213. See also, Optical paths
by class, 213
evaluation, 212–213
class 1 systems, 212
class 2 systems, 212–213
class 3 systems, 213
Optical bandwidth/electronic bandwidth, 32.
See also, Bandwidth.
Optical breakdown, 198, 680, 682, 685, 687,
703, 705.
Optical coatings, maintenance, 116.
Optical coherence tomography (OCT),
609–610.
of human retina, 609
schematic, 610
Xenopus laevis embryo, 610
Optical components, chapter, 43–59.
Optical density (OD), 71, 81, 416.
filters, 43, 49–50
Optical disks, 586.
Optical efficiency, improvements, 143–144,
216. See also, Photon efficiency.
of disk scanners, 216
of light-emitting diodes, 143–144
Optical elements, 43–58, 128, 211.
confocal light beam affected by, 211
of Köhler illumination components, 128
light beam characteristics affected by,
211
chapter, 43–58
968
Index
Optical excitation, diagram, 82.
Optical fiber. See Fiber optics.
Optical fiber, for scanning by moving fiber
tip, 213–214.
Optical heterogeneity, specimen, 22–23.
reflection, refraction, scattering, 192–197
Optical images, electronic transmission, 5–6.
Optical materials, 158, 501.
thermal properties, 158, 248–249
Optical parametric amplifiers (OPA),
100–101, 112, 114–115, 118, 124.
components, 115
table, 101
Optical parametric oscillators (OPO),
100–102, 111–112, 114–115, 118,
541, 600.
for CARS microscopy, 600
cavity dumped, to increase white light,
113
tunable, 114–115
table, 101
Optical path. of.
4Pi, confocal, 563
commercial, 566
acousto-optical device, 55
compound light microscope, 156–157
CARS, 599, 601, 907
CARV-2 disk scanner, 230
confocal, 10, 208–209, 212, 632, 681
beam-splitter, 213
disk-scanner, 12, 216
folded, 166
scanning systems, 214
fiber-optic confocal, 508
interferometers, 243, 245
Kino single-sided disk scanner, 229
LaVision-Biotec, Trimscope, 907
Leica, TCS AOBS, 910
magnetic resonance imaging, 621
Minsky confocal, 5, 25
for measuring photon efficiency, 34
multi-photon, 540, 681, 708–709
multi-focal, 552, 555
spectrometer, 424
Nikon C1si, 911
Olympus DSU disk-scanner, 230
Olympus Fluoview-1000, 912
optical coherence tomography, 610
optical projection tomography (OPT),
611
Petran tandem scanner, 228
selective plane illumination (SPIM), 613,
673
or simultaneous BSL and fluorescence,
128
surface 3D imaging, SIM/EFIC, 608
surface spherical aberration, 405–406
STED, 573
structured-illumination, 266
Visitech VT-Infinity and VT-eye, 914
Yokogwawa dual-disk-scanner, 231,
915
Zeiss LSM-510, META, 916–917
Zeiss LSM-5-Live, 50, 232, 916
Optical performance, practical tests,
652–660.
axial chromatic registration, 658
axial resolution using mirror, 656–657
chromatic aberration, 659
chromatic registration, 657–658
contrast transfer function (CTF), 656
coverslip thickness vs. RI, table, 654
field illumination, 658
flatness of field, 659
Focal CheckTM beads, 657, 658
lateral resolution, 655
resolution test slides, 655–656
specimen self-lensing artifacts, 659
spherical aberration correction, 654–655
Optical power, specimen plane, table, 140,
644.
Optical probes, 737. See also, Dyes;
Fluorescent indicators; Fluorophors;
Fluorescent labels.
Optical projection tomography (OPT),
610–613.
lamprey larva, 612
mouse embryo, 612
refractive index, 613
setup, 611
Optical pulse length, 537–538. See also,
Pulse broadening.
group delay dispersion, 537–538
group velocity dispersion, 537
measurement, 115, 901–903
Optical resonator in laser, 81–82, 116.
laser, 81–82
maintenance, 116
Optical sectioning, 9–10, 13, 180, 182, 222,
223, 236, 268–270, 469, 748,
763–764, 772, 774, 775, 784. See
also, Deconvolution, Confocal,
etc.
algorithms for widefield, 763–764
of A. Thaliana root, 772, 775
with confocal laser-scanning microscope,
9–10
example, 182, 463, 471, 492, 656
dynamic imaging, 784
improvement, with deconvolution, 752
latex bead, 3D image, 196
limiting excitation, 223
near surface of living embryo, 748
near to refractive index interface, 180
selective plane illumination, 748
structured illumination, 268–270
with widefield phase-dependent imaging,
13
Optical system, losses, 25–32, 217.
Optical transfer function (OTF), 164–165,
490–491, 562, 563, 567, 569–570,
578. See also, Point-spread function;
Contrast transfer function.
4Pi microscopy, 562, 563, 567
contrast, 164–165
deconvolution for image enhancement,
490–491
I5M, 569–570
point spread function, 490–491. See also,
Point-spread function
STED comparison, 578
Optical tweezers, 89–90, 110, 218, 383, 385.
setups for integrating, 218
table, 385
trapping wavelength, 89–90
Optics, general, 12, 125, 156–157.
finite vs. infinity, 156–157
Optiscan confocal endoscope, 213–214.
Organic dyes, 109, 203, 342–343, 353–356.
See also, Dyes; Fluorophores;
Fluorescent labels; Fluorescent
probes.
AlexaFluor, 353–355
BOPIDY, 353–355
classes, table, 355
coumarin, 353, 355
cyanine, 353–355
fluorescein, 353–355
rhodamine, 109, 203, 353, 355
Organic light-emitting diodes (OLED), 143.
Orthogonal-plane fluorescence sectioning
(OPFOS), 672–673.
Oryza sativa. See Rice.
Oscillating-fiber scrambler, 8.
Osmotic permeabilization, 359.
OTF. See Optical transfer function.
Out-of-focus light.
deconvolution vs. confocal microscopy,
461.
information, 26, 32, 487, 644–646.
Output amplifier, reconstructing analog
signal, 64.
Output modulation, of semiconductor lasers,
108.
Overheating, of filters, 43. See also,
Thermal variables.
Overlap alignment protocol, montaging,
732.
Over-sampling, 60, 70, 728.
vs. duplicate-and-smooth process, 70
reasons for, 68
subpixel, resampling, 478–479
Oxygen sensor, 45, 347.
P
Pack-and-go mode, Power Point, 842, 844.
Paeonia suffruticosa, 421.
Panda pattern, polarization-preserving fiber,
88.
PAS. See Periodic-acid Schiff.
Passively mode-locked lasers, 111.
Patch clamp, for loading dye, 360, 726–727,
734, 738–740.
Patch pipette, 738.
Pattern analysis. See Automated
interpretation of subcellular patterns.
Patterned-illumination microscopy, see
Structured illumination microscopy
PC. See Personal computer.
PCA. See Principal component analysis.
P-CARS, Polarization-sensitive detection
CARS.
Index
PCF. See Photonic crystal fiber.
PE. See Photoelectrons.
Pear (Pyrus serotina), spectrum, image,
711.
Pearson’s correlation coefficient, 668.
Pellicle beam-splitter, 216, 228–229, 231,
346.
Peltier cooling.
CCDs, 234, 447
cell chamber, 387–389
lasers, 85, 106–108, 111, 117
Penetration depth, 177, 343, 643, 672, 731,
765.
of dyes, 360, 387, 731, 739, 882, 874
of fixative, 369–370, 376, 857
FRET sensors, 798–799
long laser wavelengths, 109, 416, 418,
427–428
multi-photon, 381, 418, 433, 435, 439,
543, 545, 558, 646, 684, 708, 714,
728, 749, 904
in plant imaging, 779
in scanning electron microscopy, 847
in SPIM, 613, 675–678
TIRF, 177–178
Peony flower, autofluorescent petals,
173–174, 176, 421, 423.
Peptide-mediated uptake, 359.
Perfusion.
chambers, 381, 386–389, 394, 726, 729,
769, 870–873
fixation, 376
Periodic grating. See Grating.
Periodic-acid Schiff (PAS) reagent, 262,
369, 770, 774–775, 778.
maize pollen grain, 202
Periodically poled (PP) waveguides,
114–115.
Perrin-Jablonski diagram, 516, 517, 697,
792.
photobleaching, 697
Personal computer (PC), performance
needed for image processing,
289–290.
Perspectograph, early studies, 789.
Petrán disk, 2, 6, 11, 135, 141, 215,
223–224, 228, 251, 265, 381, 387,
447, 458, 554.
Petrán, Mojmir, 2, 6, 11, 215, 223, 228.
pH imaging, 188–189, 221, 346, 348, 359,
386, 421, 517, 529–530, 664,
739–740, 743, 744.
calibration, 421, 530, 745
display, 287
intensity image, 529, 530, 739, 740,
744
lifetime image, 530
pH indicators, 346, 739–742.
pH shift/formaldehyde fixation, 370–371,
373.
Phalloidin, as correlative marker, 235–236,
344, 376, 378, 694, 696, 756, 804,
854–856.
Pharmacological screening, 813–814.
Phase and intensity determination from
correlation and spectrum only
(PICASO), 115.
Phase contrast, 9, 171, 368, 372, 453, 506,
643, 649, 731, 851, 854, 890, 892.
coherent light for, 130
depth of field, 13
and holography, 7
scanning, 9, 13, 386
Phase fluorometry, 518–519, 526.
comparison of FLIM methods, table, 526
excitation/emission signals, 519
fluorescence lifetime imaging, 518–519
Phase randomization, to scramble light, 8,
13, 84, 131–132, 143, 507.
Phase-dependent imaging, depth of field, 13.
Phase-shifting interferometry, 245.
Phenotyping, 623–624.
Phong shading, 308–309.
Phong/Blinn models, 308.
Phosphoinositide signaling, 799.
Photo efficiency. See Photon efficiency.
Photoactivatable dyes. See Photoactivation.
Photoactivation, 187, 224, 383, 385, 541,
543–545, 693, 759.
example, 759
genetically encoded
Kaede, 187, 383, 385
Kindling, 574, 760
PA-GFP, 187, 383, 385, 752, 759–760
table, 385
Photobleaching, 174, 218, 224, 275,
341–342, 362–363, 545, 690–700,
729, 747–748, 759. See also,
Bleaching, and Chapter 39.
autofluorescence, 698
defined, 218, 691
dynamics, as a source of contrast,
202–203
effect on contrast, 174
fluorescence intensity loss, 691, 694, 696,
698+
fluorescent image of single protein, 699
fluorescent probes, 362–363
fluorescent recovery vs. irradiation time,
699
fluorophores signal optimization, 341–342
choice of fluorophore, 342
fluorophore concentration, 342
light collection efficiency, 217, 341
protective agents, 36, 341–342, 363,
368, 375, 499, 694
spatial resolution, 341
in four-dimensional imaging, 747–748
green fluorescent protein (GFP), 690, 692,
698
intentional See Fluorescence recovery
after
photobleaching (FRAP)
kinetics, 695
mechanisms, 340, 691–693
FRET, 691
multi-exponential fluorescent bleaching,
697
969
multi-photon microscopy (MPM), 545
Perrin-Jablonski diagram of bleaching,
697
photocycling, fluorescent proteins, 698
propidium bound to DNA, plot, 695
reactive oxygen species, 341–342,
362–363, 390, 544, 682–684, 691,
693–694, 852–853
reduction in, 693–696
antifade agents, 36, 341, 368, 375, 499,
694
disk-scanning microscopy, 224
quantum dots, 694
results, in living embryos, 759
of single molecules, 696–698
structured-illumination methods, 275
two-photon excitation microscopy
(TPEM), 690, 697
Photocathode, PMT, 28–29, 232–233.
quantum efficiency, 232–233
to reduce transmission losses, 28–29
Photoconductivity, in photodetectors, 252,
253.
Photocycling, fluorescent protein molecules,
698.
Photodamage. See Phototoxicity.
Photodetector. See Detectors; Light
detectors;
CCD; EM-CCD; PMT etc.
Photodiode, 134–135, 253–255, 610,
707–708.
feedback, to stabilize laser, 87, 682
feedback, to stabilize arc/filament,
134–135, 137
in hybrid PMT, 29, 30
infrared sensitive for IR lasers, 707
photometer sensor, air space, 26
quadrant, for alignment, 87, 134
of self-aligning source, 134–135
for testing display software response, 830
vacuum avalanche, 254, 255
Photoelectric effect, and LED operation,
137.
Photoelectrons (PE), 29, 30, 62–63, 77,
232–234, 254–255, 257, 259–264,
339, 633, 863.
amplification of, 62–63
in the CCD, 232–234, 495, 918, 931
production in PMT, 30
single-PE pulse-height spectrum, 29, 77
secondary electrons, as source of PMT
multiplicative noise, 77
Photoemissive devices, 256–257.
Photoemissive effect, 254.
Photographic recording systems, 6–7,
11–12, 20, 22, 30, 71–72, 132, 139,
141, 162, 207, 217, 263, 280, 488,
581+, 588, 590–591, 593–594, 607,
613, 628–629, 633, 640, 643, 712,
829, 862, 865–867.
“toe” response, quadratic, 71
Photometer paddle, to measure light beam,
26, 35, 139–140, 159, 391, 650–651,
665.
970
Index
Photometric response, and HD curves, 71.
Photomicrography (Loveland), 139.
Photomultiplier tube (PMT), 9, 28–31,
35–36, 51, 62–63, 74–75, 222, 232,
251, 254, 255, 258–261, 443, 527,
661–662.
after pulsing, 257
Bio-Rad, 260–261
as confocal detectors, pros/cons, 222
for epi-fluorescence confocal microscope,
9
functioning, 62–63
GaAs photocathode, 28–29, 232, 252,
255, 263, 527, 931
gain from collisions at first dynode, 31
grey levels, 443
hybrid, single-pixel signal levels, 31,
254–255
linearity, 661–662
microchannel plate, 232–233, 255, 262
mini-PMT arrays, 51, 667
multiplicative noise, 28–30, 77, 633,
677, 926. See also, Multiplicative
noise
in multi-channel detection systems, 51
noise and gain, 74–75
number of photons striking per unit time,
35–36
optical enhancer to increase QE, 28–30
photon counting, 21, 29–30, 32–35,
258–259, 260–263, 542
quantum efficiency, 527
vs. cooled CCD, 26–28
signal variation with time, 232
transit time spreads, 527
Photon(s), 20–21, 30, 33–36, 63–64, 132.
counting precision, 20–21
uncertainty, 63–64
interactions with photomultiplier tube, 30
lost, 33–36
Photon counting, 21, 29–30, 32–35,
258–259, 260–263, 542.
circuits, 33–34, 258, 521
digital representation of optical data,
32–33
effects, 34–35
examples, 35, 263
hybrid PMT, 29–30
pile-up losses, 32–33, 35, 78, 521, 523,
526–527
with PMT, 29–30, 32–35, 258–259, 260,
263
Photon detector types. See Detectors and
entries by each detector type.
CCD, 254
direct effects, 252
image dissector, 254–255
microchannel plate, 232–233, 255, 262
MCP-CCD, 262
gated, 519, 523–524, 527, 532
photoconductivity effects, 252, 253
photoemissive, 254
photovoltaic, 252–253
thermal effects, 252
vacuum avalanche photodiode, 254, 255
work functions, table, 252–253
Photon efficiency, 24–36, 215, 217, 341,
631.
defined, 24
as a limitation of confocal systems, 24,
223
measuring, 26, 33–36, 217
practical confocal microscopy, 631
of scanners, 215
table listing photon losses, 217
Photon flux, statistics, 256–258.
Photon interactions, 252–256.
Photon (shot) noise, 660–661. See also,
Poisson noise.
Photonic crystal fiber (PCF), laser delivery,
1, 88, 109–110, 113, 504, 541.
for white light source, 113
Phototoxicity, 112, 363–364, 390–391, 651,
729, 746, 770.
chapter, 680–689
in brain slices, 729
damage is higher to either side of raster,
54
factors influencing, table, 363
fluorescent probes, 363–364
live cells, 390–391
reduction, 391
from uneven scan speed, 651
Photo-uncaging, 187, 210, 383, 385, 541,
544–545, 692, 729, 760, 912. See
also, Photoactivation.
Photovoltaic effect, 252–253.
Phycobiliproteins, 338, 341, 343, 355–357,
693.
Physical limitations, 20, 24, 63–64.
on accuracy and completeness of data,
20
Poisson noise, 63–64. See also, Poisson
noise
Physiological fluorescence imaging, 383,
385.
PICASO. See Phase and intensity
determination from correlation and
spectrum only.
Piezoelectric effect, defined, 57.
Piezoelectric focus controls, 166, 215, 219,
222, 231, 241, 245, 268, 468, 754,
909.
Piezoelectric scanning systems, 57, 215,
238, 510, 555, 610.
Piezoelectric devices.
AOD driver, 54–55, 57
acousto-optical components, 54–55, 57
to align objective, 166
dithering to increase CCD resolution,
70
effect described, 57
to focus objective, 166, 215, 219, 222,
231, 241, 245, 268, 468, 754, 909
laser alignment, 87
light scrambler, 84
to move optical fiber, 84
to move scanning mirror, 57, 215, 238,
510, 555, 610
to move stage, 215, 567
phase-shifter
in 4Pi confocal, 609
in structured illumination, 268
optical coherence tomography,
609–610
stretching optical fiber, 609
Pile-up, of pulses.
in avalanche photodiode, 253
in photomultiplier tube output, 32–35
measuring risk of, 34–35
p-i-n diode, 253.
Pinhole, 26–28, 33–35, 149, 150, 154, 201,
210, 213, 215, 224–228, 395,
631–632.
advantages and disadvantages, 26–28
calibrating diameter, 33–34
confocal, proper use, 28
disk-scanning, 224–228
mini-image detection, 32
optical fiber as, 506–507
optimal size, 226–227, Chapter 22
Fraunhofer formula, 225
position in confocal microscope, 210
practical, in confocal, 631–632
radius, effective, 35
ray paths, different sizes, 226–227
single-mode polarization preserving fiber,
213
small pinholes, effect, 225
of tandem scanners, 215
vibration shifts relative positions, 201
Pinhole disks, critical parameters, 224–228.
Pinhole energy, with spherical aberration,
149, 150, 154, 631–632.
penetration into water, 149, 150
defocus and NA, 150
defocus and wavelength planapochromat,
154
Pixel clock, digitization, 62, 64–65, 201,
234–235, 258, 903, 923, 929.
CCD, table, 929
Pixels, 38–39, 60, 62–63, 65, 258–259.
defining, 60
digitization, 62–63
optimal, 63–64, 66
representing intensity, 258–259
and resolution, 38–39
and Abbe criterion resolution, 38–39, 65
PKzip, 580.
Plan objectives, Zeiss, field diameter, table,
152.
Planapochromat, 152, 155. See also,
Objectives.
flatness of field and astigmatism, 152
lateral chromatic aberration, 155
Plancks law, energy of photon, 35, 137, 252,
424.
Planar illumination, SPIM, optical
sectioning, 751.
Index
Plane of focus, distortion, 16, 23.
by beam deviations, 16
by refractile cellular structures, 23
Plant cell imaging, 769–785.
autofluorescence, 770–772
birefringent structures, 162–164.
420–421. See also, Birefringence
chamber slides for plants, 429
clearing intact plant material, 166,
417–418, 420, 439, 610, 624,
774–775
computer visualization methods, 778
deconvolution, 784–785
direct imaging, 772–773
dynamic imaging, 783–784
effect of fixation, 195, 428
Equisetum, 774
fluorescence properties, 421–428
emission spectra, 421–423
microspectroscopy, 421–426
fluorescence resonance energy transfer,
425. See also, FRET
harmonic generation See Harmonic
signals fungi, 438–439
genetically encoded probes, 769, 773,
783
green fluorescent protein fusions, 773,
783
of green tissues, 770
hairs, 434–435
history, 769
light attenuation in plant tissue, 414–418
A. thaliana, example, 416
absorption spectrum, 415
effect of fixation, 428
maize stem spectra, 417, 418
M. quadrifolia spectra, 416
M. quadrifolia optical sections, 419
Mie scattering, 162–163, 167, 417–418
nonlinear absorption, 416–417
Rayleigh scattering, 162–163, 167, 417,
703
light effects on, 770
light-specimen interaction, 425–428
living plant cell specimens, 429–439
calcofluor staining procedure, 424,
438
callus, 429
cell walls, 168–169, 188–189, 303,
306, 416–417, 420–421, 428–431,
435–136, 438, 439, 710–711,
713–715, 769–776, 779–781
chamber slides, use, 429
cuticle, 434–437, 715, 717, 779
fungi, 438–439, 624, 782, 870
hairs, 431, 434–436, 772
meristem, 168, 420, 430, 770, 776–778,
783
microsporogenesis, 431–432
mineral deposits, 163, 420, 436–438,
703
pollen germination, 420, 433–434, 781,
783
pollen grains, 202, 305, 313, 420,
431–433, 553, 558, 781, 783
protoplasts, 195–196, 203, 416, 421,
423–427, 429–431, 438–439, 693
root, 172, 174, 303, 307, 421, 429,
430+, 438, 464–465, 556, 772–773,
775, 777, 779–783
culture chamber, 429
starch granules, 202, 420–421, 428,
432–433, 435, 703, 710–712, 715,
719
stem, 168, 172, 180, 417–419, 421,
424, 429, 556, 707, 710–711,
713–714
storage structures, 435–436
suspension-cultured cells, 189,
429–430
tapetum, 433–434, 779
waxes, 420, 428, 434–435, 714–715
new spectral tools, 770
obtaining spectral data, methods, 772
penetration values, 779
photodamage, 770
point spread function, 722, 784
refractive index heterogeneity, 192,
418–420
single-photon confocal excitation,
772–778
specific methods, 769
spectral unmixing, 770
examples, 665–666
staining, 774
technological developments, 769
textbooks, 769
three dimensional, 771
clearing agents, 166, 417–418, 420,
439, 610, 624, 774–775
deconvolution protocols, 784
reconstruction, 775–776
segmentation, 776–778
two-photon excitation, 415–419, 421,
423
advantages, 778–779
best conditions, 781
compared with one photon, 421
cell viability, 779–782
deconvolution protocols, 784
dyes, 782
green fluorescent protein, 782–783
light-specimen interaction, 425–427
microspectrometer, 424
pitfalls, 782
thick specimens, 779
in vivo, 781
Plasma membrane, microscopy. See Total
internal reflection microscopy
(TIRF).
Plasma light sources, spectra, 44.
Plasmid DNA, nick-damage, 684, 724,
802–804. See also, Microinjection;
Electroporation; Biolistic
transfection.
Plasmodesmata, 777.
971
Plumbago auriculata, fluorescence spectra,
422.
PMT. See Photomultiplier tube.
p-n diode, 253. See also, Photodiode.
PNG (Portable network graphic), 581, 584.
Pockels cell, variable beam attenuator, 25,
54, 57, 87, 116, 543, 701, 903–904.
Pockels effect, in crystals, 57.
Point-spread function (PSF), 4, 10, 23, 27,
39, 68–70, 145–146, 189–190, 208,
223, 239–250, 271, 275, 330, 378,
405, 407, 409, 446, 448, 453–457,
485–486, 489–494, 536, 562–564,
570, 574, 578, 635, 656, 674, 750,
784, 830, 895.
3D, 68–70, 247–248
4Pi microscopy, 562–563
additional information from, 570
space invariance of PSF for, 564
apodization, 240, 243, 249, 250, 272, 567,
889
blind deconvolution, 468, 485
in botanical specimens, 772, 784
in brain slices, 729
calculations, RI-mismatch, 407
for glycerol, table, 409
for water, table, 409
CARS, 596
comparing widefield with confocal, 27,
453–457, 493, 644–647
confocal, 10, 12, 208+, 212, 216, 405,
632, 681
vs. deconvolution, 27, 453–457, 493,
644–647
deconvolution, 189–190, 223, 489,
490–494, 784. See also,
Deconvolution
quantifying PSF, 492–494
deformation caused by RI anomalies,
22–23
Fourier transform, 489, 490
lateral resolution. See Lateral resolution
measuring, 240–242, 455, 462, 471,
656
amplitude/phase, 242
fiber-optic interferometer, 240–241
images, 246–248
high-NA objectives, 239–250, 492, 656
pupil function, 240
for 3D deconvolution, 145–146
non-linear, 552, 750
and Nyquist, 635, 636, 751, 752
optical transfer function, related to,
490–491
polarization effects, 249–250
pupil function, 245–248. See also, Pupil
function
Rayleigh-criterion and Nyquist sampling,
39
refractive index mismatch, 405, 407
spherically aberrated, 148–150, 407, 492
shape in telecentric systems, 208
SPIM, 674
972
Index
Point-spread function (PSF) (cont.)
STED, diagram, 574, 578
structured illumination see Structured
illumination microscopy
temperature effects, 25, 85, 248–249, 630
terminology, 405
Wiener filtering, 494
Points, defined, 59.
Point-source, for measuring photon
efficiency, 33.
Poisson noise, 20–21, 29, 37, 63–64, 67, 69,
74–75, 81, 164–165, 211, 232, 234,
442, 456, 460–463, 468, 487, 495,
497, 633–636, 647, 651, 655, 660,
693, 784, 835, 923–924, 926. See
also, Quantum noise, Shot noise.
bleaching, 693
of CCD
charge transfer, 920
dark charges, 921–922
CT imaging, 615
and display linearity, 72–73, 588
digitization, as part of signal, 65, 69,
633–636
of EM-CCD, 233–235, 262, 927–928
and FLIM, 524–525
and gray levels, 74
importance of deconvolution, 38–41, 60,
69, 189–190, 222–223, 320, 399,
471–472, 481, 495, 751–753, 835
intensity spread function, 75–78
photomultiplier tube, 74–75
affects effective QE, 31
multiplicative noise, 29, 647, 660
in photon detection, 63–64
and pixel size, 64, 68, 633–636, 928
practical effects, 67
single-pixel noise, 65, 67, 190, 635, 832,
835–836
spectral unmixing, 667, 770
examples, 665–666
structured illumination, 278
uncertainty in contrast, 74, 164–165
and visibility, 37, 667
Polarization, 13, 49, 57, 83, 88, 89,
211–212.
attenuator, 43, 543, 907
beam-splitter, 13, 50–51, 57, 85, 87, 100,
171, 217, 513, 631, 904
to avoid spectral distortion, 49
circular or phase randomized, 211–212,
229
effect on AODs, 55
effect, of dichroic beam-splitters, 34,
49–50
Kerr cell, 111, 113, 516
of laser light, 8, 83, 88–89, 113, 478,
558
optical components, 57, 155, 211
optical fibers, 213
Pockels cell, 25, 54, 57, 87, 116, 543,
701, 903+
rectified DIC optics, 846
to reduce reflections, 6, 25, 141, 158, 171,
516, 229. See also, Antiflex system
scramblers, 8, 84, 132, 143
Polarization effects, 211, 249–250, 503.
birefringence, 188, 420–421, 431, 434,
436, 438, 480, 503. See also,
Birefringence
blind deconvolution, 479
and CARS microscopy, 595, 600–604
high-NA objective lenses, 249–250,
267
interaction with nucleus, 23
optical fibers, 503, 507
stereo image displays, 299, 589
Polarization microscopy, 43, 50–51, 154,
156, 162, 188, 288, 348, 438,
479–480, 513, 555, 711, 714–715,
717, 719, 891, 894.
centrifuge microscope, 8
of collagen fibers, 164, 188, 717
DIC, 10, 14, 127, 146, 468, 473
and FRET, 793
and harmonic generation, 179, 428,
704–706, 717, 719
MFMP, 555
mitotic apparatus, 15, 717
p- and s-, and incidence angle, 50–51
Pol-scope, 13, 188, 432, 468, 480
PSF, 406–407
to regulate light intensity, 43
STED, 578
Polarization noise, in lasers, 83.
Polarization-preserving fiber, 49, 87, 503,
505, 507.
as a pinhole, 213
Polarization-sensitive detection CARS (PCARS), 600, 601, 604.
adipocyte cells, 604
Polarized light, 7, 14, 83–85, 146, 158, 162,
171, 229, 406–407, 420, 479, 894.
deconvolution, 479
image formation, 406–407
PSF, 479
Polarizer, 83, 128, 188, 249, 268, 275, 420,
479, 711, 903–904.
for antiflex, 6, 84, 141, 158, 229
for attenuation, 43, 85, 87–88, 543,
903–904
for CARS, 601
Glan-Taylor, 85, 87, 100, 171
Glan-Thompson, 85, 904
LCD, 589, 715
micro-wire, 85
structured illumination, 264
tunable, 715
Pollen germination, 433–434.
Pollen grains, 202, 305, 431–433, 438, 553,
556, 558, 678, 781, 783.
germination, 433–435, 783–784
multi-focal multi-photon imaging, 556
Pol-scope, 13, 188, 432, 468, 479–480
test specimen, 195, 269, 313, 553, 556,
678
Pol-scope, 13, 188, 432, 468, 479–480.
deconvolved, 479
images, 15, 188, 479, 717
Portable network graphic. See PNG.
Position, accuracy in CLSM, 40.
Position sensors, galvanometer, 53–54.
Posterizing, 591.
Potassium titanium oxide phosphate (KTP)
crystal
for non-linear optical frequency conversion,
107.
Potato (Solanum tuberosum) SHG signal,
712.
Power requirements, for lasers, 65, 80–81.
Power spectrum. See Contrast transfer
function.
Power supply, laser as noise source, 86.
PP, Periodically poled waveguide,
114–115.
Practical confocal, 2-photon microscopy,
tutorial. See also, each topic as a
major entry.
2-photon
excitation duty cycle, 644
peak power level, 644
photodamage vs. penetration, 645
power vs. penetration, 646
3D microscopy methods compared, table,
647+
best 3D method for, 644–647
biological reliability, 631
bleaching pattern, 627–628
quantum efficiency, 628
chapter, 627–649
confocal images with few photons, 634
deconvolution, factors, 646
filling back-focal plane, 210, 509, 629,
633
focus, compensating drift, 395, 732
getting a good confocal image, 629–631
alignment of optics, 629–630
back-focal plane (BFP), 210, 509, 629,
633
focus, 629
low signal, 631
mirror test specimen, 630
no signal, 631, 660
simultaneous BSL/fluorescence,
631
getting started, 627
Köhler illumination for transmission, 34,
127–128, 131, 229, 627, 648–649
multi-photon vs. single-photon, 646
new controls, 631–636
biological reliability, 631
pinhole size, 631–632
pixel size, 62, 634–635, 784, 928
Nyquist reconstruction/deconvolution,
635–636
over-sampling, 635
photon efficiency, 24–26, 215, 217, 341,
631
pinhole summary for, 26–28, 633
Index
pixel size, 62, 634–635, 784, 928
measuring, 635
summary for, 636
poor performance, reasons, 640–643
air bubbles, 643
curvature of field, 641
dirty objective, 642–643
imaging depth, 643
under filling objective pupil, 642
optical problems, 640–641
sampling problems, 640
singlet-state saturation, 643
under-sampling, 635
schematic diagram, 632
statistical considerations, 633–634
stray light, 201, 632, 904
test specimen, 636–640
description, 636–637
diatom, 638–640
figures, 637–640
reasons for, 636
widefield vs. beam scanning, 647
Prairie Technologies, LiveScan Swept Field
design, 237.
Pre-amplifier, in digitizing analog signal,
64.
Precompensation, in fiber optic cables, 88.
Presentation software, 829–845.
helpful URLS, 844–845
movies, 837–844
artifacts, 839–840
coding limitations, 838
compression of large movies, graph,
843
compression of PAL TV movies, table,
842
digital rights management, 844
entropy, 841
frame count matching display cycle,
838–839
MPEG display formats, 840–841
overlaying, 844
Pack-and-go mode, 842, 844
performance benchmarks, 841–842
region code, 844
remote use, 842–844
rules 837–838, 844
up-sampling, 838–839
very high resolutions, 841
precautions, 829–830
testing, 830–836
aliasing gallery, 834
aligning images, 835
brightness, 832
changing display size, 832–835
codecs, 831
compression, 835–836
compression artifacts, 837
cropping, 835
digital rights management (DRM), 830
down-sampling in PowerPoint, 834
fast graphics cards, 831, 832
gamma, 832–833
measuring display speed/sensitivity,
830
random color dot image, 836
reference images, 830–831
removing distortion, 835
resolution, 832–835
rotating, 835
scaling, 835
screen capture, 830
static image performance, 831
step image, 833
under-sampled image, 835
up-sampling, example, 834
viewer, 830
Preventive maintenance, lasers, 115–116.
Principal component analysis (PCA),
731–732.
Printers, 591–593.
aliasing, 592
color images, 592
grey levels, 592
ink jet, 593
laser, 593
posterizing, 591
scaling techniques for, 592
Prionium, MMM image, 556.
Probe, mismatch with pixel shape, 39.
Processor performance, 3D-image display,
289+.
Projection/compositing rules, 3D-image
display, 302–304, 763–764.
alpha blend, 302, 304
average intensity, 302
first or front intensity, 302
Kalman average, 304
maximum intensity, 302
Propidium iodide, 344, 355, 360, 426,
651, 693–695, 773, 778–779, 782,
812.
dead cell indicator, 426, 651, 875, 877
Proteins, 195, 756, 760, 794–795, 804. See
also, Green fluorescent protein, etc.
chimeric fusion, 794
fluorescent, FRET, 794–795
Kaede, 187, 383, 385
Kindling, 760
microinjection, 804
PA-GFP, 187, 383, 385, 752, 759–760
tagged, 756, 758
translational fusions, 756
UV absorption, 195
Proteomics, 237, 790, 804, 809, 818, 867.
location, 825
Protoplasts, 195, 416, 429, 430, 431.
A. thaliana, 195–196, 203, 416, 421,
423–427, 429–431, 438–439, 693
Proximal tubule, labeled, 744.
Pseudo color display, 173–175, 190, 291.
PSF. See Point spread function.
Pulse broadening, 88, 111, 210, 537–538,
543, 606, 609, 728, 903.
Pulse length measurement, 115, 901–903.
Pulse spreading. See Pulse broadening.
973
Pulsed lasers, 81, 96–100, 110–114, 120,
137. See also, Lasers; Ultrafast
lasers.
broadband tunable, table, 120
diode, table, 96–97
DPSS, table, 98
dye, table, 96
excimer, table, 96
for FLIM, 537
kits, table, 98, 100
nitrogen, table, 96
scanning only region of interest, 237
for 2-photon excitation, 81
ultrafast, table, 99–100
vapor, table, 97
Pulse-counting mode, 21, 29–30, 32–35,
258–259, 260, 263.
Pump sources, for dye lasers, 103.
Pumping media, maintenance, 116.
Pumping power vs. frequency cubed, 65, 82.
Pupil function, 211, 245–248.
3D point spread function restored,
247–248
4Pi, 566–567
AOD, 56
empty aperture, 248
of human eye, 72, 128
intermediate optics, 211, 222, 225, 250
Köhler illumination, 34, 127–128, 131,
229, 251, 627, 648–649
Mach-Zehnder interferometry, 245
measurement, 246–248
images, 246–248
objective, 24, 155, 158–159, 211,
239–240, 242, 492, 551–552, 554,
566–567, 650
orthonormal Zernike polynomial for,
table, 247
phase-shifting interferometry measuring,
245
polarizing effects, 249
pupil plane, 50 See also, Back-focal plane
transfer lens, 728
view of pupil image, 629
Zernike polynomial fit, 245–247
Purkinje cells, Golgi-stained, 167–168.
Pyrus serotina. See Pear.
Q
QE. See Quantum efficiency.
Q-switched pulsed laser systems, 111,
114–115.
Quantitative analysis, flying-spot
microscope, 6–7.
Quantization, limitations imposed by, 37–39.
See also, Chapter 4.
Quantum dots, 221, 343, 357–358, 360–361,
656, 694, 696, 757, 801, 814, 846,
853. See also, Semiconductor
nanocrystals.
assays for, 814
in electron microscope, 852–854
FRET, 801
974
Index
Quantum dots (cont.)
labeling, 853
toxicity, 357, 694
Quantum efficiency (QE), 25–30, 74–78,
222, 232–234, 238, 251, 254–255,
349, 355, 375, 383, 390, 442–443,
459, 516, 527, 575, 628, 646, 556,
703, 751, 793, 920–922.
of back-illuminated CCD, 77–78
charge-coupled device (CCD), 26–28,
74–76, 142, 215, 232, 234, 257–258,
261, 644, 707, 751, 754, 810,
920–921
comparative among CCD cameras, 76
effect on Poisson noise, 74–75
effective QE, of photon detectors, 28, 29
of electron-multiplier CCD, 4, 30, 59,
234, 920
FLIM, 516–517, 520, 523, 526–527, 529,
530
FRET, 792
of human eye, 251
and intensity spread function, 74–75
and multiplicative noise, 77
optical enhancer, to increase QE, 28–30
optimal 3D microscopy, 644
photomultiplier tube (PMT), 26–28, 51,
77, 222, 257, 262, 527, 707
graph, 29
table, 707
signal-to-noise ratio, 263, 442–443
variation with wavelength, 29
vs. wavelength, 922
Quantum noise, 21 63–64, 69–70, 468, 472.
See also, Poisson noise.
and approximation, for reconstruction,
69–70
Quantum wells, as absorbers, 111.
Quantum yield, of fluorescent dyes, 172,
180, 338–345, 353, 360, 363, 383,
421, 543–544, 574, 661, 683, 690,
710, 737, 792, 794–795.
Quartz-halogen lamp, control, 138–139. See
also, Halogen lamps.
R
Rabbit, 237, 744.
antibodies, 855, 877–878
kidney proximal tubule, pH, 744
Radiance, of non-laser light sources, 126,
132, 137–139, 141.
measuring with radiospectrometer,
139
Table, 1140
Radiospectrometer, radiance vs. wavelength,
139.
Raman background, in glass fibers, 88, 90,
162, 506–507.
lower in large-mode-area, fiber, 110
Raman scattering, 162–163, 167, 339–340,
348, 506–507, 545, 697.
and bleaching, 697
defined, 162
Raman spectroscopy, 48–49, 90, 167, 254,
339–340, 507, 545, 697. See also,
CARS.
CARS, 204, 550, 577, 595–605
chemical imaging, 90
hard-coating on interference filters used,
48–49
image contrast, 167
Ramp-up, for light sources, 136, 137.
and long-term stability, 137
and short-time stability, 136
Rare earths, for doping fiber lasers, 110.
Raster, 62–64.
convolution, 485–486
dimensions, in specimen, 63
retrace, 25, 33, 53–54, 219, 338, 389,
543, 628, 651, 908. See also,
Retrace, raster scanning shape,
63
size, vs. pixel size and light dose, 64
temporal limitations, 141
Raster scanning, 5–6, 25, 141–142, 223,
540, 596.
alignment, 629–630, 651
assymmetrical sampling, 38–40
bleach pattern, 3D, 538, 628, 693
chromatic aberration limitations, 156,
640–641
damage is higher to either side of raster,
54
display, 830–831, 835
distortion, 40
and electronic bandwidth, 70, 238
for fast confocal imaging, 223
fiber-scanning, 214, 508
galvanometer limits, 52–54, 223, 651
limitations imposed by AODs, 56
MPEG formats, 840
Nyquist sampling, 38, 41, 59–60, 62,
634–635
off-axis aberrations, 151, 640–641,
659–662
pattern on Nipkow disk, 5–6, 223–225.
See also, Nipkow disk scanning
retrace gating, 25, 54, 56, 219, 389, 543,
628, 651, 908
scan angles, 209, 214
stability, 708
sampling in time and space, 141–142
timing, 33, 53, 753
zoom, raster size and magnification, 11,
24, 37, 63–64, 66, 70, 79, 317, 389,
493, 627, 634–636, 655–658, 683,
731
Rat, cells and tissues, 205, 320, 323, 330,
398, 739, 813.
brain slices, 393, 398, 686
CA1 region, 323
cardiac muscle, 498, 529, 556
cerebellar granule neurons, 813
EDL muscle, calcium, 740
fixation, 370, 372, 393
hippocampus, 268, 317, 341
interossi muscles, SNARF-1, pH image,
739
intervertebral disk, 310–311
kidney, 511, 803
leukemia cells, 347, 520–521
FLIM image, 521
neuron, membrane potential, 205
tooth, 667
Rate, imaging, limited by signal level, 73.
Ratiometric imaging, 189, 346–347. See
also, Calcium imaging, pH, etc.
bleach ratio, 697–698
calcium, 736–737, 850. See also, Calcium
imaging
CARS, 600, 602, 604. See also, CARS
concentration calibration, 742–745
to detect colloidal gold labels, 167
to determine ionic concentration, 36
FLIM, 516–532. See also, FLIM
FRET, 174, 184, 790, 794–795, 797–798.
See also, FRET
glutaraldehyde autofluorescence assay,
369
HCS, high-content screening, 813,
823–824.
indicator choice, 738
interpretation, 740–741
live/dead assay, 875
pH, 739–744. See also, pH imaging
structured illumination. See Structured
illumination microscopy
water-immersion objectives, 737
Rayleigh criterion (Abbe criterion), 1–3, 9,
37–39, 60–61, 66, 129, 146, 486,
703, 822, 928.
breaking the Abbe/Rayleigh barrier,
571–573
Nyquist sampling, 39, 60, 66
of two point images, 1–3, 146
Rayleigh scattering, 162–163, 166, 167,
339, 342, 417, 703, 747.
compared to Mie scattering, 163
in embryos, 747
by colloidal gold labels, 167
light attenuation in plant tissue, 417
wavelength dependency, 162–163
Rayleigh unit, 147.
Reactive oxygen species (ROS), 341–343,
362–363, 390, 544, 682–684, 691,
693–694, 852–853. See also,
Bleaching; Phototoxicity.
as basis of correlative TEM staining,
852–853
Readout noise, 74–75, 77, 232. See also,
Noise.
and readout speed, 77
Real image, disk-scanners, 224.
Real-time 2D imaging, 12–13, 167–168,
215, 222–224, 232, 235, 307, 496,
542.
Real-time 3D imaging, 154.
Receptors.
cholera toxin, 790–791, 796–797, 802
Index
deconvolution, 495
EGF, 533
ERD2, 791, 796
fibrinogen, 846–847, 850
high-content screening, 809, 812–814
KDEL, 790, 797
ligands, 354
lipid, 790, 791
proteins, 357
Streptococcus, 879
transferin, 819
uncaging, 545
Reconstruction, 3D.
definition, 280.
Nyquist and filtering/deconvolution, 59,
66–67, 69, 70, 173, 235–236,
280–315, 458, 468–469, 474–475,
496–497, 563, 585, 603, 607, 610,
615, 635, 672, 675, 677–678, 690,
772, 730, 731, 762, 77, 774–776,
778, 784, 883
Recording times, 141–142.
in widefield microscopy, 141–142
using LED source systems, 141–142
Recovery curve, after bleaching, 187.
Red fluorescent protein (RFP), 221–222.
Reference list.
historic, 889–899
lasers, 123–125
Reflected-light images, 180, 181. See also,
Backscattered light.
confocal, of integrated circuit, 180
of glass bead, in water, 181
Reflecting objectives, constraints, 156.
Reflection contrast technique, Antiflex, 159.
Reflection mode, low coherence light, 130.
Reflectivity, optical surfaces, 159, 163,
167–171.
anti-reflection coatings, 158
on-axis, artifact, 168–171
refractive index, 159, 163, 167
Refracting regions affect imaging beam,
15–16.
Refractive index, (RI), 14–15, 23, 45, 148,
152, 163, 198, 377, 404–413,
418–420, 613, 654. See also,
Spherical aberration; Dispersion.
anomalies in, effect on PSF, 23, 418–420
of biological structures, 163, 377
table, 277
of botanical specimens, 418–420
coverslip thickness, importance, table,
654
of immersion medium, 277, 411
effect on PDF, 23, 418–420
effect on sharpness, 14–15
effect of wavelength and temperature
on, 148, 248–249, 411
and intensity, and spectral broadening,
111
of layers in interference filters, 45
of mounting media, table, 198, 342,
370–371, 373–377
of optical glass vs. wavelength, 152
optical projection tomography (OPT), 613
self-shadowing, 198
temperature, 148, 248–249, 411
of tissue/organs, table, 377
Refractive index mismatch, effects,
404–413. See also, Spherical
aberration.
table for glycerol, 409
table for water, 409
calculation, 404–407
dependence of focal shift, 410
diagram, 404
dry objectives, 410–411
experiments, 409–410
water/glycerol results, table, 410
field strength calculation, 405
other considerations, 410–413
spherical aberration correctors, 15, 151,
147, 192, 411–412
terminology, 405
actual focal position (AFP), 405
focal shift, 405
nominal focal position (NFP), 405
theory, 404–407
Region code, for MPEG-encoded movies,
844.
Region-of-interest (ROI), 835.
brain slice, 726, 733
diagonal, 658
display presentation, 835
embryos, 747, 759
FRAP, 51, 187
FRET, 797, 801
in image processing, 289, 300, 323, 330,
676
labeling, 353
must be smaller at high resolution, 577
nanosurgery, 219, 686
photobleaching, 690
preprocessing, 676
rapid acquisition, 236–237
structured illumination, example, 272
viability studies, 683
Registration synthesis method, 328–331.
defined, 328
landmark-based, 328–329
multi-view deconvolution, 330
Relationships, in fluorescence microscopy,
80.
energy per photon, 80
flux per pixel, 80
photons/s vs. wavelength, 80
Relative motion, objective vs. specimen,
39–40.
Relaxation, in laser energetics, 82.
Relay optics (telan lenses), 145, 157, 214,
455.
Reliability.
of 3D image, 461, 517
biological, vs. damage, 24, 68, 631, 633
lasers, 80, 102, 115
living cell work, 387
975
mirror position, 40
photometric, 312
spectral detectors, 662
Removable storage media, 585–588.
random-access devices, 586–588
compact disks (CD), 586–587
digital video disks (DVD), 587–588
floppy disks, 586
magnetic disks, 586
MO (magneto-optical) disks, 586
optical disks, 586
WORM (write once, read many) disks,
586
Rendering, of 3D views, 280, 285, 290, 301,
307, 309, 311, 377, 749, 762, 764.
definition, 280
voxel speed, 290
RESOLFT microscopy, 571–574, 577. See
also, STED.
breaking diffraction barrier, 571–573
concept, 571–573
different approaches, 573–574
ground state depletion (GSD), 573
STED, 573–574
outlook for, 577
resolution, new limiting equation, 571
triplet-state saturation, 573
Resolution, 1, 4, 13, 16, 24, 36–41, 59, 61,
65–67, 210. See also, PSF; FWHM.
adequate levels, 36–41
axial, 13
axial-to-lateral ratio vs. NA, 4
back-focal plane diameter, table, 210
confocal vs. non-confocal, 16
and contrast transfer function, 37, 59, 61
estimating, 65–67
measured, widefield, 16
minimum resolvable lateral spacing, 1, 16
spatial and temporal, 24
sufficient, 36–37
Resolution, structured illumination.
Fourier-space, 270–271
linear image reconstruction, 271
Lucosz’s formulation, 273
methods, 270–276
Moiré effects, 270–271
photobleaching, 275
reconstruction results, 272
standing-wavefield microscope, 275
thick samples, 274, 275, 278–279
Resolution scaling, STED comparison, 578.
Resolution test slides, 16, 656.
Resonant cavity, laser, 81–82, 111, 115.
Resonant scanners, 52–54, 56–57, 223, 447,
543.
acceleration distorts mirror shape, 53
blanking, 25, 218, 338, 389, 543, 628,
651, 908
compared to acousto-optical deflector, 56
duty cycle, 52
galvanometer, 52
multi-photon excitation, 543
raster-scanning, 33, 53–54, 56
976
Index
Resonant scanners (cont.)
retrace, 54, 56. See also, Retrace, below.
scan speed, 54
Retrace, raster scanning shape, 25, 54, 56,
219, 389, 543, 628, 651, 908.
acousto-optical deflector, 56
blanking, 25, 219, 338, 389, 543, 628,
651, 908
raster-scanning, 33, 53–54, 56
Review articles, listing, 889.
RFP. See Red fluorescent protein.
Rhodamine, dyes, 81, 109, 116, 136, 140,
203, 264, 292, 339, 342–345, 353,
355, 362–363, 375–378, 409, 538,
553, 592, 693, 697–698, 762,
783–784, 794, 851, 854–856.
arsenical derivatives, 348
bleaching, 697, 698
calibration plot, 661, 851
excitation of, 181, 109
fluorescence correlation spectroscopy,
693
FRET, 347
photobleaching quantum yield, 363
planar test specimen, 538
power for 1-, 2-photon excitation, 81, 3
41
Rhodamine-123, 374, 389
resolution measurement, 409
stability and cost, 116
Rice (Oryza sativa), 168, 171, 414, 415,
712, 715.
absorption spectrum, 415, 706
backscattered light image, 168, 171
emissions spectra, autofluorescence, 713
leaf fluorescence images, 714–715
light attenuation in plant tissue, 414
silica deposits, 714–715, 717
Richardson-Lucy, deconvolution, 497, 568.
Richardson Test Slide Gen III, 652, 656.
RLE. See Run-length encoding.
RNA, microinjection of, 803, 804.
RNA labels, 344, 369, 465, 531–532, 612,
691, 758, 874–875.
ROI. See Region of interest.
Room light, as stray signal, 201, 632, 904.
Roots, plants, 172, 174, 303, 307, 421,
430–432, 438, 464–465, 556,
772–773, 775, 777, 779–783.
maize, image, 432
mounting, 429, 431
ROS. See Reactive oxygen species.
Rose Criterion, 37–38, 68, 164, 633.
relationship with signal-to-noise ratio,
164
for visibility, 37
Rotating, specimen, 188, 568, 835.
micro-CT, 615
optical projection tomography, 610–611
SPIM, 672–673, 676, 751
Rotor, galvanometer, detecting position,
53–54.
Run-length encoding (RLE), 580.
S
Safety, 83, 85, 90, 115, 117–118, 124,
132–139, 900, 903, 904.
arc sources, 132–139
beam-stop design and use, 118. 903–904
classification of laser systems by hazard,
117
cleaning objectives, 642
display geometry, 297
equipment needed, 900
eye protection
against Brewster surface reflections, 83
goggles, 118
with external-beam prism method, 90
fiber optics for transporting laser light,
88
hazardous materials
fluorescent laser dyes, 85, 103, 116
used beryllium oxide tubes, 115
high pressure Xe lamps, 136
monitor power to avoid explosions,
138–139
in disk-scanning confocal microscope,
231
laser, 117–118, 839, 900, 903–904
installation requirements, 85
monitor power to avoid explosions,
138–139
references, list, 123
safety curtains, 117, 904
training, 118
SAM, saturable absorber mirror, 111.
Sampling. See Digitization, 20, 63–64.
non-periodic data, 38
optimal, 63
Saponin, formaldehyde fixation, 359, 375,
856.
Saturable absorber mirror, pulsed lasers,
111.
Saturable Bragg reflector (SBR), 111.
Saturable output coupler (SOC), 107, 111.
Saturation, singlet-state fluorescence, 21–22,
41, 142, 265, 276, 339, 442, 448,
450, 643, 647, 899.
performance limitations, 81, 450, 928
SBR, saturable Bragg reflector, 111.
SBT. See Spectral bleedthrough.
Scaling techniques, 592, 835.
Scan angle, and position in image plane,
209–210.
Scan instability, detecting, 40–41.
Scan raster, testing, 651–654.
malfunctioning system, 653
phototoxicity from uneven scan speed,
651
sources of fluorescent beads, table, 653
well-calibrated system, 652–653
x and y galvanometers, 651–652
z-positioning calibration, 652, 654
stability, 652
Scanned-slit microscopes, table, 224.
Scanner arrangements, evaluation,
213–215.
Scanners, 51–55, 57, 214–216.
acousto-optical deflectors, 55. See also,
AODs
mirror arrangements, 214
evaluating, 215–216
mechanical, 51–54. See also,
Galvanometers
piezo-electric, 57, 215, 238, 510, 555,
610
single mirror/double tilt, 215
sinusoidal, “tornado” mode, SIM scanner,
52
Scanning electron micrographs, 428, 434,
437, 846–848, 850–852.
Scanning laser ophthalmoscope (SLO), 480.
Scanning fiber-optical microscopy. See
Fiberoptic confocal microscope.
Scatter labeling for tracing lineage, 461,
462.
Scanning systems for confocal light
microscopes. See also,
Galvanometers; Disk-scanning
confocal microscopy; Acoustooptical deflectors; Linescanning
confocal microscopes; Raster.
Lissajous pattern, circular scanning. 554
“tornado” mode, SIM scanner, 52
Scattering, 162–163, 167–171, 550.
coherent anti-Stokes Raman (CARS), 550
elastic, Rayleigh, 162–163, 166–167, 339,
342, 417, 703–747
Raman, 162, 167, 339–340, 348,
506–507, 545 and reflection contrast,
167–171
Scattering object, viewed by TIRM 177. See
also, Backscattered light.
Schiff reagents, 262, 369, 770, 774–775,
778.
Schottky diode, photodetector, 253.
Scientific thought, four aspects, 789–790.
Scion Image, 281–282, 395, 730.
Scramblers, light, 8, 13, 84, 131–132, 143,
507.
Screen capture, 830.
Screens, to enclose laser beams, 118.
SD. See Static discharges.
SDA. See Stepwise discriminant analysis.
Sea urchin, S. purpuratus, 173, 198, 200.
Second harmonic generation (SHG), 90,
114–115, 166–167, 179, 188, 550,
552, 556, 703–719, 729–730. See
also, Harmonic signals.
as autofluorescence, 361
cell chambers, 166, 429, 552
detectors, 706–708, 728
disk-scanning, 552, 556
double-pass detection, 166–167
table, 706–708
crystals for SHG, 103, 107, 114–115, 188,
703
energy relations, 705
in lasers, 103, 107, 114–115
layout, 166, 191, 552, 708–709, 712
Index
light attenuation spectra, 706
light sources, 706–708
brain slices, 729–730
non-linear optical microscopy, 704–705
optically active animal structures in,
714–717
brain slice, 729–730
collagen structure, 703, 717
sarcomeres, 716
spindle in mouse zygote, 717
spindle in zebrafish embryo, 718
structures producing SHG, table, 715
table of structures, 715
zebrafish embryo, 716, 718
optically active plant structures, 428,
710–714
Canna, nonlinear absorption, 710
cell wall, 428, 711, 714
Commelina communis, 712
emission spectrum of maize, 710, 711
Euphorbia pulcherrima, spectrum, 710
mineral deposits, 436
Pyrus serotina, spectrum, 711
rice leaf, 712, 715
starch granules, 433
maize, 710–711, 713–714
emission spectrum, 710–711
leaf spectrum, 710
pol-microscopy, 711
stem, optical section, 714
stem, spectrum, 710, 713
chloroplasts, tumbling, 713
membranes of living cells, 90
mineral, deposits, 436
photodetector suitability, table, 706–707
polarization dependence, 71, 717–720
potato, as SHG detector, 712
pulsed laser suitablity, table, 706
signal generation, 179, 552, 597, 704–705
spectra, 706
spectral discrimination, 421
starch granules, 433
Segmentation, FLIM image, 527–528.
Segmentation methods, 281, 283–285, 290,
300–302, 304–306, 309, 311–312,
316–319, 321–330, 333–334,
527–528, 776–778, 812.
3D, 776, 822, 828
automated, 818, 821–822, 828
background, 321
blob segmentation example, 322–324
gradient-weighted distance transform,
323
model-based object merging, 323–325
watershed algorithm, 323–324
bottom-up, 321
combined blob/tube segmentation,
328–330
foreground, 321
hybrid bottom-up/top-down, 322
integrated, 322
intensity threshold-based, 321
object, 321
for plant cells, 774–777
balloon model, 776
watershed algorithm, 322–325, 777,
822
region-based, 321–322
top-down, 322
tube-like object segmentation, 324–328
mean/median template response, 328
skeletonization methods, 324–325
vectorization methods, 324–327
validation/correction, 333–334
manual editing, 333–334
Selective plane illumination microscopy
(SPIM), 613, 614, 672–679, 751.
3D scanning light macrography, 672
anisotropic resolution, 678
applications, 675
axial resolution, 674–675
vs. CLSM, 678
Drosophila embryogenesis, 675–676,
747–748, 751–752, 754, 756, 759,
804, 810
and FLIM, 527
images processing, 675–678
image fusion, 676–677
pre-processing, 676
registration, 676
lateral resolution, 674
light-sheet illumination, 672–674
light sheet thickness, 674–675
Medaka, 614–615
heart image, 614
embryo image, 675
multi-view reconstruction, 675–678
point spread functions (PSF), 674
schematic setup, 613, 673
thin, laser light-sheet microscope,TLLSM,
672
Self-aligning arc source, 135.
Self-shadowing, 165, 174, 194, 195.
in confocal optical sections, 174
spherical structure, 195
in epi-fluorescent mode, 165, 194
SEM. See Scanning electron microscope.
Semi-apochromat, pros and cons, 158.
Semiconductor lasers, 86, 105–108.
noise sources, 86
Semiconductor nanocrystals (quantum dots),
221, 343, 357–358, 360–361, 656,
694, 696, 757, 759, 801, 814, 846,
853.
as probes, 221, 757, 759
Semiconductor saturable absorber mirror
(SESAM), 107, 111.
for self-starting intense optical pulse
trains, 111
Sensitivity, video photodetectors, 6–7.
Sensitized emissions, of acceptor, 795–796.
See also, FRET.
Sequential devices, 585–586.
Serial sampling, single-beam confocal, 20.
SESAM, Semiconductor saturable absorber
mirror, 107, 111.
977
SFP. See Simulated fluorescence process.
Shannon, Claude, 64–65.
Shannon sampling frequency, defined, 64,
443.
SHG. See Second harmonic generation.
Shift invariance, deconvolution, 457, 490,
564.
Short-pass filters, 43–44.
Shot noise, 232, 256–257, 286, 442,
460–461, 495, 558, 660–661, 831.
See also, Poisson noise, Quantum
noise.
Signal, 27, 62. See also Speed relationship
to magnification, 62
Signal attenuation-correction, 320–321.
Signal detection, basics, 660–663, 918–931.
See also, Detectors.
coefficient of variation, 660
instrument dark noise, 660
photon (shot) noise, 660–661
PMT linearity, 661–662
signal-to-noise ratio, 660
spectral accuracy, 662
spectral resolution, 662–663
wavelength response, 663
Signal levels, 16-photon peak signal, 73–74.
Signal-to-background ratio, of titaniumsapphire laser, 112.
Signal-to-noise (S/N) ratio, 37, 53–54, 67,
81, 164, 251, 257, 265, 330, 340,
386, 391, 442–451, 470, 481, 495,
498–499, 528, 542, 562, 567, 582,
599, 621–622, 660, 690, 696, 699,
707, 736–737, 740, 753, 769, 772,
778–780, 810, 813.
3D imaging, 448–451
4Pi microscopy, 562–567
bleaching, 391, 442, 690, 696
in calcium imaging, 737
chapter, 442–451
comparative performance, 256, 448–451
bleaching-limited performance,
448–450
configurations of microscope, 448, 449
disk-scanning microscope, 449
line illumination microscope, 449
saturation-limited performance, 450
scanning speed effects, 53, 450–451
structured illumination, 265–266, 270,
275–276, 279–280
S/N ratios for, table, 450
widefield (WF) microscope for, 450
confocal microscope, 444–447, 660
calculations, 444
detectability, 446–447
methods compared, 450
noise model N1, 444–445
noise model N2, 446–447
deconvolution, 470, 481, 495, 498–499
designs, confocal, 212–216, 447–448, 450
disk-scanners, 221
dynamic range, 2-photon, 644, 778–780
high-content screening, 810
978
Index
Signal-to-noise (S/N), ratio (cont.)
improvements, 736
micro-CT, 615
magnetic resonance microscopy, 621–622
multi-photon fluorescence microscope,
112, 427, 447, 542, 779
Nyquist sampling, 67, 448
optimal excitation power, 81, 340
Rose criterion, visibility, 37–38, 68, 164,
633
saturation, 442
vs. scan rate, 53
signal level, 67, 75, 528
sources of noise
background noise, 443–444
grey levels, 443
quantum efficiency, 442–443
shot noise, 442–443
sources of noise, 442–444
STED, 574
and visibility, 37
Silica glass, transmission losses, 502.
Silicon diodes, near infrared emission, 132.
Silicon-intensified target (SIT) camera, 730.
brain slices, 730
SIM. See Surface imaging microscopy.
Simplicity, as design goal, 43, 66, 220, 229,
387, 508, 647.
Simulated fluorescence process (SFP), 310.
Single-cell automatic imaging, 809, 812.
Single-cell calcium imaging, 812.
Single-longitudinal-mode fiber laser, 110.
Single-mirror/double tilt scanner, 215.
Single-molecule, 80.
biochemistry, 221–222, 575, 690, 693,
696
bleaching, 690, 693, 696, 697–698, 699
Single-photoelectron pulse heights, 30.
Single-photon, energy, equation, 35.
Single-photon counting avalanche
photodiodes (SPAD), 527.
Single-photon excitation, plant imaging,
772–778.
Single-photon pulses. See Photon counting.
Single-scan images measure scan stability,
40–41.
Single-sided disk scanning, confocal
microscopy, 132, 141–142, 168, 171,
175, 215–216, 229, 231, 907, 913.
See also, Disk-scanning confocal
microscopy.
advantages and disadvantages, 215–216
basic description, 141
commercial, 907, 913
light source, 132, 141–142
Singlet state saturation, 21–22, 41, 81, 142,
265, 276, 338–339, 442, 448, 450,
643, 647, 899, 928.
Sinusoidal bidirectional scanning, 25,
52–54. See also, Resonant scanners.
duty cycle, 53, 260
Sinusoidal image, 831, 838.
fiber-optic confocal, 510
Sinusoidal modulation, in FLIM, 524–526.
SIT. See Silicon-intensified target camera
imaging.
SLF. See Subcellular location features.
Slice AM-dye-painting protocol, 726–727.
Slice chamber protocol, 727.
Slit scanning confocal, 12, 25, 37, 50, 51,
56, 221–226, 231+, 235, 238, 519,
522, 664, 741, 914, 916.
Achrogate, 50, 212, 231–232, 916
with AOD scanning, 56, 914
commercial, 913–914, 916
critical parameters, 224–228
optical sectioning, 228, 444–449
optimal slit size, 225
point excitation, slit detection, 914
SLM. See Spatial light modulator.
SLT. See Subcellular location tree.
Smart media, digital storage, 588.
SMD. See Surface mount device.
SNARF-1, 345, 346, 531, 739, 744–745.
ratiometric pH label, 744–745
stained rat interossi muscles, 739
table of variants, 531
Snell’s law of refraction, 167, 654.
SOC. See Saturable output coupler.
Software packages, visualization, table,
282–283.
SoftWorx, 3D display software, 282.
Solanum tuberosum, potato, 712.
Solid state memory devices, 588.
compact flash cards, 588
memory stick, 588
smart media, 588
Solid-state photodetector, 30–31, 918–931.
See also, CCD; EM-CCD.
Solid-state lasers, 86, 103–118, 236–237.
cooling, 108
noise sources, 86
thin-disk lasers, 109
tunability, 109
use, 236–237
Source brightness, measure, radiance units,
126.
Source optics, reflecting and collecting light,
134.
Space invariance, telecentric systems,
207–208.
Space multiplexing, in MMM, 555.
Spacer, material in interference filters, 46.
SPAD, single-photon counting APD, 527.
Spatial coherence, 84.
Spatial filter, 89, 107, 391, 542, 708, 729.
optical devices for, 89, 222–223, 729
digital, 391–392. See also, Gaussian
filtering
Spatial frequency, 37, 60, 65, 66. See also,
CTF.
and contrast transfer function, 37
and geometry, 66
response of microscope, and pixel size,
65
zero, as measure of brightness, 60
Spatial laser beam, characteristics, 89.
Spatial light modulator (SLM), 266.
Spatial orientation factor, for FRET,
792–793.
Spatial resolution, in confocal microscopy,
24. See also, Resolution, PSF, CTF.
Special setups, for CLSM, 218–219.
Specifications, general, for scanner, 54.
Specimen, general considerations, 192–197,
228, 361–362, 779. See also, Living
cells, Living embryo imaging.
fluorescent probes interactions, 361–362
cytotoxicity, 362
localization, 361–362
metabolism, 361–362
perturbation, 362
optical heterogeneity, 22, 23
plants. See Plant cell imaging; Botanical
specimens
Specimen chambers. See Living cell
chambers.
Specimen heating, in 2-photon, 539.
Specimen holder, for scanning specimen, 9.
Specimen preparation, for automatic 3D
image analysis, 319–321.
image analysis, 319–321
imaging artifacts, 320
Specimen preservation, general, 368–378.
antibody screening on glutaraldehydefixed specimens, 377
evaluation, 371–374
cell height to measure shrinkage,
371–373
defined structures, distortion, 373–374
MDCK cell, stereo image, 373
MDCK cell, vertical sections, 372
fixation/staining, 370–371
fixative characteristics, 368–370
chemical fixatives, 369
cross-linking fixatives, 369
freeze substitution, 369
microwave fixation, 369
protein coagulation, 369
formaldehyde, 369–370, 373
general notes, 374–378
glutaraldehyde, 369, 370
immunofluorescence staining, 371
improper mounting, 376
labeling thick sections, 376–377
microwave fixation, 377–378
mounting methods, 370–374
critical evaluation, 371–374
mounting media, table, 377
pH shift/formaldehyde fixation, 370–371,
373
refractive index mismatch, 377
mounting media, table, 377
refractive index of tissue/organs, table,
377
tissue preparation, 376
triple labeling, 375–376
Specimen-scanning confocal microscope,
9.
Index
Speckle, from high-coherence sources, 8,
84, 90, 130–132, 448.
Speckle microscopy, 13, 383, 385, 889.
Spectra, emission.
arcs, 130
black body, 136
LEDs, 133
solar, 127
tungsten source, 153
Spectral accuracy, 662.
Spectral bleedthrough (SBT), 185, 203–204,
664.
in intensity-based FRET, 185
Spectral confocal image A. thaliana
seedling, 175.
Spectral detector, 203–204, 662–663,
666–667.
testing, 662
Spectral discrimination, filter bandwidths,
44.
Spectral imaging, 175, 382, 384.
table, 384
Spectral leakage, inter-channel signal
imbalance, 185, 203–204.
Spectral phase interferometry, for direct
electric field reconstruction
(SPIDER), 115.
for pulse length measurement, 115,
901–903
Spectral properties, of filters vs. angle, 49.
Spectral resolution, of detection system,
203–204, 662–663, 666–667.
Spectral response.
of CCD chips, 29, 234, 922
of eye, 153
PMT photocathodes, 29
Spectral transmission, objectives, plots,
159–161.
Spectral unmixing, 190–192, 319, 361, 382,
384, 386, 423–425, 431, 664–667,
770, 905.
detectors for, 51, 667
examples, 665–666
limitations, 51, 382, 667
overlapping fluorophore emission, 190,
319, 423–425, 664–667
removing autofluorescence using, 667
Spectrofluorimetry, for FRET, 793, 795.
Spectroscopic ruler, 765.
Speed, in confocal imaging, 7, 11–12, 36,
41, 53, 142, 222–224, 235–236, 434,
447, 450, 458, 460, 482, 526, 536,
563–564, 748, 753–755, 784. See
also, Temporal resolution.
4Pi-MMM, 563–564
AOD, 55–56
calcium imaging, 741
CARS, 599–600, 604
charge-coupled device cameras, 77–78,
142, 229, 231–235, 259, 647, 651,
754–755, 885
data compression, 581–582, 586–588
detector, in FLIM, 523, 558
disk-scanning confocal, 141, 216, 224,
754
for display, processing, 803, 839, 841842, 862
factors affecting, 235–236, 482, 496,
753–754
of fixation, 370
FRET, 795, 805
galvanometer, 52–54, 211, 214
high-content screening, 809–810, 813
MMM, 551–555, 563–564
need for, in living cell imaging, 222,
753–754
rendering, 3D display, 831
SPIM, 613, 678
Spermatocyte, crane fly, 15.
Spherical aberration, 15, 34, 147–149, 151,
160, 192–197, 208, 241, 244, 247,
330, 395, 404–413, 454–455, 463,
466, 471, 480–481, 542, 629, 640,
654–655, 657–658, 728, 772, 774,
893, 903–904. See also, Aberrations,
spherical.
blind deconvolution, 471, 480–481
chapter, 404–413
confocal microscopy performance, 654
correction of RI mismatch, 192, 287, 411,
542
correction of, figure, 145, 411–412,
654–655
corrector, 92, 395, 398, 411, 477, 640,
654
deconvolution, 463, 466, 468–469, 471,
480, 498–499, 658, 728, 784
effect of specimen, 192–197, 418, 454,
747
index mismatch. See Index mismatch
measurement, 145, 407, 455, 471,
481, 492, 657
signal loss, 330, 389, 395, 413, 457, 661
SPIDER, Spectral phase interferometry for
direct electric field reconstruction,
115, 901–903.
Spill-over, between detection channels. See
Bleedthrough.
SPIM. See Selective plane illumination
microscopy.
Spinning disk, 3, 5–6, 11, 40, 141, 176, 216,
223–224, 231–232, 235–236,
260–265, 459–460, 464, 468,
481–483, 783–784, 810–811. See
also, Diskscanning confocal
microscopy.
commercial, 907, 913, 915
FLIM, 519–520, 522
high-content screening, 810–811, 820
MMM, 554, 558
performance, 449–450
systems for, cytomic imaging, 810
vs. TPE imaging, in plant cells, 783
Yokogawa CSU-10/22, 231. 915
Spinning-disk light scrambler, ground glass,
8.
979
Spinning filter disk, digital projector, 590.
Spirogyra, and depth of optical sections,
195.
Spot scanning, to avoid coherence effects,
84.
Spot size, full-width at half-maximum. See
Pointspread function, Full-width
half-maximum.
Square pixels, advantage of using, 62.
Stability, 86, 102, 103, 136–139, 826.
algorithmic, 473
arc sources, 136–137, 477
argon-ion laser vs. krypton laser, 102
disk scanners, 215
of DVDs, 587
dye. See Dyes; Bleaching from fiber-optic
coupler, 505–506
galvanometer, 54
halogen sources, 136–139, 346
interferometer, 240–241, 267
laser, 81, 85–89, 704
diode, 106, 108–109
fiber output, 505
helium-cadmium, (low), 103
intensity, 85–87, 113, 116, 136, 477,
903
measurement, 650–651
pointing, 87, 903
results, 86, 103
structure, 82–85, 103
thermal, 111
wavelength, 106–108, 115, 118
mechanical, 39, 82, 85, 201, 267, 512,
652
objectives, 146
photostability, 363, 369, 690–702, 802.
See also, Dyes; Bleaching
scan, 40, 638–639, 651
shutter, CCD camera, 929
thermal, 111, 219, 387, 389, 394, 539. See
also, Thermal variables
Stage-scanning confocal microscope, 11.
piezoelectric scanners, 57, 708
Staining, plants, 438, 774. See also, Dyes;
Livingcells; Botanical specimens;
Plant cell imaging; Fluorophors.
calcofluor procedure, 438
of plant tissues, 774
Standards, ISO (DIN) microscope design,
156+.
Standing-wavefield microscope, 275.
Starch granules, plant, 202, 420–421, 428,
432–433, 435, 703, 710–712, 715,
719.
Static discharges, destroy semiconductors,
109.
Statistical noise, in counting quantummechanical. See Poisson noise.
STED. See Stimulated emission depletion.
Stem-cells, 623, 678, 762, 790, 813.
Stem, plant, 168, 172, 180, 417–419, 421,
424, 430, 556, 707, 710–711,
713–714.
980
Index
Stentor coeruleus, backscattered light image,
168.
Step index optical fibers, 501–503.
Stepwise discriminant analysis (SDA), 818,
820.
Stereo Investigator, software, 282.
Stereology, 316, 319.
Stereoscopic image, about, 6–7, 9, 11, 154,
224, 298–299, 317, 396.
biofilms, 880
cheek-cell specimen, 23
diatom, 640
Drysophila, microtubules, 752
embryo, 200
fat crystal, polarization, 479
neurons, 298, 314
Alexa stained, 330
backscattered light images, 167
eye, optic nerve, 481
Golghi-stained, 298
Lucifer-yellow, 314
microglia, 396–398
rat-brain neurons, 398
transmitted light, 475
lung, 292
MDCK cells, 373–374, 378
Milium chromosomes, Fuelgen-stained,
298
Paramecium, chromosomes, 298
pea root, RNA transcript, 465
platelet, high-voltage, EM, 848–849
sea urchin, S. Purpuratus, 173, 198, 200
skin, 298
Spirogyra, 195
tandem-scanning confocal microscope,
6
Stereoscopic views, image processing and
display, 290, 292, 293, 295–299,
451, 764.
color space partitioning, 297
display, 293, 299
interlaced fields of frame, 297
movie projection, 838
pixel-shift/rotation stereo, 297
stereo images example, 298
synchronizing display, 297
Stick objective, for in vivo confocal, 806.
Stimulated emission depletion (STED)
microscopy, 3, 539, 561, 568,
571–578.
axial resolution increase, 576
breaking the diffraction barrier, 571–573
challenges, 577
compared to confocal, 575–576
diagram, 573
different approaches, 573
dyes used successfully, table, 575
OTF compared to confocal, 578
outlook, 577
PSF compared to confocal, 578
RESOLFT, the general case, 572–573
results, 576, 578
triplet-state, 573
Stimulated emission of radiation, defined,
82–83, 124.
Raman scattering, 167
semiconductor, 106
and stimulated-emission depletion, 573,
577
STN, supertwisted nematic, 589.
Stochiometry, ion kinetics, 741.
Stokes field intensity, 595, 597.
Stokes laser, in CARS microscopy, 595,
597–604.
Stokes shift, 44–45, 268, 338, 341, 343,
443–447, 539, 542, 690, 759,
792–793.
anti-Stokes,
CARS, 550, 595–604
defined, 44–45
in fluorescence resonance energy transfer,
792+
large, in 2-photon, 539, 646
of quantum dots, 694, 759
size of fluorophores, 45
Storage, digital. See Data storage.
Storage structures, plant, 435–436.
maize, image, 436
Stray light, 58, 632, 904.
laser light, 632
non-descanned detection, 904
practical confocal microscopy, 632
room light, 201, 632
Streak camera, FLIM detector, 520.
Strehl ratio, measure of image sharpness,
247.
S. purpuratus (Sea urchin), 173, 198, 200.
embryo, 173, 198, 200
first mitotic division, 173
image degradation, from top and
bottom, 198
stereo-pairs of embryo, 200
Structural contrast, 188. See also, Harmonic
signals.
Structure, optical, 59, 68, 132–135.
of light-emitting diodes (LED), 133
of microscope sources, 132–135
recognizing features in noisy images, 68
chapter, 265–279
Structured illumination microscopy,
265–279.
advantages/disadvantages, 265
computing optical sections, 268–270
vs. confocal microscopy, 265
degree of spatial excitation modulation,
268–270
absolute magnitude computation,
268–269
homodyne detection scheme, 268–269
max/min intensity difference, 268
scaled subtraction, 269–270
square-law detection, 268–269
synthetic pinholes, 268, 269
experimental considerations, 265–268
illumination masks for, 266
light source for, 267
pattern generation, 266–268
schematic setup, 266
nonlinear, 276
resolution improvement, 270–276
Fourier-space, 270–271
linear image reconstruction, 271
Lucosz’s formulation, 273
Moiré effects, 270–271
photobleaching, 275
reconstruction steps/results, 272
standing-wavefield microscope, 275
test results, 274
thick samples, 274, 275, 278–279
Subcellular location features (SLF) in
automatic image analysis, 819–820,
822–824, 828.
2D, 819–820
2D SLF feature descriptions table, 819
3D SLF, 822–823
test results, table, 824
Subcellular location tree (SLT), 825.
Subpixel deconvolution, 478–479.
Subresolution beads, 655–656. See also,
Beads.
Sun, microscope light source, 126–127, 131,
135.
spectrum, 127
Superficial optical sections, living embryo,
748.
Supertwisted nematic (STN), 589.
Surface imaging microscopy (SIM),
607–608.
mouse embryo, 608
setup, 608
Surface mount device (SMD), for LED,
133.
Surface orientation, affects reflected light,
181.
Surface structures, distortion, 197.
Surface topography, maximum intensity,
180.
Surfaces, of interference filters, 47.
Suspension-cultured cells, 189, 429–430.
bacteria, 876, 878
image, 430
frozen, 854
Swept-field confocal microscope, 238.
Synchrotron, wide-spectrum light source,
135+.
Synthetic pinholes, in structuredillumination
microscopy, 268, 269.
images, 269
SYTO, 396, 874–876, 879–885.
T
Tagged image file format. See TIFF.
Tandem-scanning confocal microscope
(TSM), 2–6, 11, 13–15, 39–40, 141,
167, 215–216, 223–224, 228–229,
447.
comparison with other confocals,
13–15
Index
description, 6, 141, 215–216, 228–229
development, 5–6
evaluation, 215, 216
observing ciliate protozoa, 141
rate of data acquisition, 11
real-time imaging of tooth, 167
sources of vibration, 39–40
viewing color/depth-coded, real-time,
stereo
images, 154, 304
Tapetum, plant, 433, 434, 779.
TEC, Thermo-electrically cooled, see Peltier
cooling.
Telan systems, 129, 157.
Telecentric plane, 208–209, 211.
conjugate, 208–209
effect of angular deflection in, 211
Telecentricity, 207, 214.
of closely-spaced scan mirrors, 214
defined, 207
Tellurium oxide (TeO2), for use in AODs,
55
TEM. See Transmission electron
microscope.
TEM. See Transverse electromagnetic
modes.
Temperature, 29, 56, 133, 135–136, 856,
885. See also, Thermal variables.
Temperature tuning, of diode lasers, 108.
Temperature effects on high NA objectives,
248+.
Temporal aliasing, 39, 41, 391, 836–837,
839.
Temporal coding, 299–300.
Temporal coherence, 7–8, 82–85, 131.
defined, 84
Temporal dispersion, 502. See also, Pulse
broadening.
Temporal displays, 292–293, 297, 836.
Temporal experiments, biofilms, 885–886.
Temporal pulse behavior, pulsed laser, 111.
See also, Pulse length measurement;
Pulse broadening.
Temporal resolution, 12, 24, 36–38, 41,
221–222, 322, 334, 386, 391, 399,
458, 558, 577, 618, 620, 622, 651,
667, 730, 737, 746, 772, 784, 801,
809. See also, Fluorescence lifetime
imaging (FLIM).
of photodetectors, 263
Temporal signals, 162, 286, 331, 383.
“Test drives,” for living embryo imaging,
752.
TFT. See Thin-film transistor.
Tetracysteine, labels, 221, 348, 359, 853.
Thalamocortical slice protocol, 724.
Thermal lensing, pulsed lasers, 109, 113,
543.
Thermal variables, 219, 856.
active medium, lasers, 81
of AODs, 56–57
arcs, peak emission wavelengths, 129
automated confocal imaging, 810
cell chambers, 117, 386–389, 394, 727,
790, 810, 814, 885–886. See also,
Cell chambers cooling, 108, 133
cryo preparation for EM, 856–857
on detectors, 29, 252, 256–257, 495
drift, 16, 115, 219, 386, 567, 489, 652
compensating, 396, 732
on dye labeling, 359, 361, 738–739
effects of anti-bleaching agents, 694
effect on bleach rate, 696–689
effect fiber pinhole size, 506
fiber-optic, pol-preserving fiber, 503
filament spectra, 135–136
fixation, 369–372, 375, 377
incandescent lamp emission, 135–136
laser cavity, 34, 82, 85–88, 107, 109, 111,
541
of LED, 133, 136–138
brightness, 133
lensing, in pulsed lasers, 109, 113, 543
and light-source output, 136, 138, 650
noise signal, 254, 257, 232–234,
261–262, 495, 660, 734, 921, 924,
925
on objective lenses, 248–249
in photography, 71
properties of ice, 856
properties of optical materials, 158,
248–249
and photomultiplier tube, (PMT), 29
on refractive index, 15, 56, 145, 411
immersion oil, 148–149, 248–249,
411
retinal exposure, 117–118
sensors, 255–256, 727
solid-state laser, 86, 108
specimen damage, 84–85, 139, 685
specimen heating, 539, 545, 681, 685,
904
temperature tuning, laser, 108, 115
thermomechanical effects, 685
time constant, 38
Thermo-electrically-cooled, see Peltiercooled.
diode lasers, 85, 107–108, 111, 117
THG. See Third harmonic generation.
Thick samples, 274, 275, 278–279. See also,
Living embryo imaging; Brain slices;
Biofilms.
background, 278
structured illumination, 274, 275,
278–279
close focus region, 279
distant focus region, 279
in focus region, 278
number of collected photons, 279
Thin disk lasers, 109–110.
Thin Laser Light Sheet Microscope
(TLLSM), 672. See also, SPIM.
Thin-film transistor (TFT), 589.
Third harmonic generation (THG), 90,
166–167, 179–180, 188, 428, 435,
550, 705–718.
981
CARS, 596–597
contrast mechanism, 166–167
deposits no energy, 361
detectors for, 421, 706–708
table, 707
double-pass detection method, 166–167
intracellular inhomogeneities tracked,
90
light attenuation spectra, 706
light sources, 706–708
to make more laser lines, 109, 114
mechanism, 705
microspectroscopy, 421
MMM, 551, 559
non-linear optical microscopy, 705
optical sectioning, 704
optically active animal structures,
714–717
collagen mat, polarization microscopy,
717
mouse zygote spindle, 717
structures producing THG, table, 715
zebrafish embryo, 716, 718
optically active plant structures, 710–714
cell walls, 438
Commelina communis, 712
Euphorbia pulcherrima spectrum, 710
maize, emission spectrum, 710, 711,
713
maize, polarization microscopy, 711
maize, stem section, 714
phytoliths, polarization microscopy,
720
potato, 712
Pyrus serotina spectrum, 711
rice leaf, image, 712, 715, 719
photon interactions, 179
pulsed lasers suitable, table, 706
STED, 577
structural contrast, 188
Three-decibel point (3dB), for bandwidth,
59, 65.
Three dimensional cell pellet, 815.
Three dimensional microscopy, 766, 771,
804+.
future perspectives, 804–805
living embryos, 766
of plant cells, 771
Three dimensional projections, embryo, 763.
Three dimensional segmentation, plant,
776–778.
Three-channel confocal microscopy.
with 4 recombinant proteins, 190
assays for, 814
Three-dimensional diffraction image, 4, 147,
407, 455, 463, 471, 491.
Three-dimensional micro-array assays,
815–816.
Three-dimensional reconstruction, 775–776,
778, and Chapters 14 and 15.
plant imaging, 775–776
A. thaliana, 778
Equisetum, 774
982
Index
Three-photon excitation (3PE), 88, 415, 447,
535, 550–552, 555, 558, 647, 680,
709, 876.
absorption cross-section, 680
damage, 682, 686
fiber-optics, 507
resolution, 447
setup, 708–709
TIFF (Tagged image file format), 580.
Tiled montage, 851, 858.
Tiger, ECDL laser system, 90.
Time correlated single-photon counting
(TCSPC), 518, 520–523, 526.
for lifetime imaging, table, 526
FLIM, 520–523
FRET-FLIM, 186
schematic diagram, 521
Time multiplexing, of adjacent excitation
spots, to reduce flare in MMM,
553–554.
Time-gated detection, FLIM, 522–524, 526,
528+.
diagram, 522
FLIM methods compared, table, 526
FLIM, image, 528–529
Time-lapse imaging, 136, 222, 354,
382–384, 392–399, 652, 773,
885–886.
Amoeba pseudopod, 191
confocal of plant cells, 773
high-content screening, 812
illumination stability, 136
image analysis, 286, 320, 333, 732–733
mechanical stability, 219
microspectrometry, maize damage,
424–426
rectified-DIC, of platelets, 846
SPIM, 613
table, 384
three-dimensional plus time, 222
two-dimensional plus time, 222
Time-lapse recordings.
Amoeba pseudopod, 191
Ascaris sperm, 846
biofilms, 885
brain slices, 725, 727, 729, 732–733
embryos, 676, 749, 752, 759, 761
meristem growth, 430
plant roots, 781, 784
rectified-DIC, of platelets, 846
two-photon microscopy, 10
TIRF. See Total internal reflection
fluorescence.
TIRM, 177–179, 477.
Tissue specimens, introducing the probe, 360.
Titanium:sapphire laser (Ti : Sa), 82, 84–86,
88–91, 94, 100–103, 105, 107, 109,
111–112, 114, 123–124, 165, 346,
358, 415, 423–424, 459, 541, 550,
551, 645–647, 688, 706–708, 713,
727, 750, 756, 759. See also, Lasers,
titanium : sapphire and Ultrafast
lasers.
4Pi, 563–564, 567
brain slices, 731
CARS, 599
compare to other fast lasers, 112–113
Cr : Fosterite, femtosecond pulsed laser,
109, 114, 415, 541, 706–709,
712–714
embryos, 750, 756, 759, 731, 764
emission stability, 86
four-level vibronic model, 82, 109
maintenance, 116
multi-photon excitation, 541
and OPOs, 114–115
plants, 415, 423–424, 706–708, 713–714,
717, 781–783
popular models, specs, table, 120
STED, 575
ultrafast, 112–113
URLs, 124
TLB. See Transmitted light bright-field.
TLLSM. See Thin Laser Light Sheet
Microscope.
Tobacco, 116, 189–190, 430, 693.
smoke, not around lasers!, 116
suspension-cells,
birefringence, 189–190
GFP expressing cells, 430
photo-bleaching, 693
“Toe” photographic response, defined, 71.
Tornado mode, SIM scanner, 54.
Total fluorescence signal, 742.
Total internal reflection fluorescence
microscopy (TIRF), 90, 160,
180–184, 223, 477, 801.
blind deconvolution, 477
vs. confocal image, 184
contrast, 180–184
cytoskeleton, image, 183
FRET, 801
limits excitation to single plane, 223
objectives, for epi-TIRF, 161
Total internal reflection microscopy (TIRM),
177–179, 477.
blind deconvolution, 477
evanescent wave generation, 178
TPE. See Two-photon excitation.
TPEM. See Two-photon excitation
microscopy.
Trade-offs, 36, 68, 78–79, 221, 224,
644–648, 747–748, 825.
beam power, visibility/damage, 693
blind deconvolution, 483, 488, 499
compression algorithms, 581, 840
confocal endoscopes, 508
when digitizing, 68, 78–79
embryo specimens, 747–748
high-content screening, optimal
clustering, 825
living cells, 381, 693
micro-CT, dose/resolution, 616
MRM, time/resolution, 622
and pinhole size, 265, 267
processing speed/segmentation, 301
speed, S/N, sensitivity and damage, 221,
224, 232, 556, 644–648
SPIM, resolution and number of views,
613
Transcriptional reporters, embryo analysis
and, 748, 755–756.
FluoroNanoGold, 854
mRNA, 316–317, 465
plants, 773, 781
NF-kB, 814
Transfection buffer, electroporation, table,
802.
Transfection, cellular, 756–758, 790, 791.
brain slices, 722, 724–725, 730–731
Transfection reagents, for chromophores,
358, 360, 362, 556, 682, 790–791,
795, 803.
2-OST-EGFP, 566
COS7, 693
EB3-GFP, 183
for FRET, CFP/YFP, 795–796, 798,
801–802
GaIT-EGFP, 566
GFP-MusculoTRIM, 184
ligand binding, 348
Transfer function, implications for image
contrast, 164–165. See also, CTF.
Transient permeabilization, 359, 373, 375.
Trans-illumination, absorption contrast, 166.
Transistor-transistor logic (TTL), 259.
Transit time spreads (TTS), 527.
Translational fusions, 756, 757. See also,
Transfection agents.
subcellular specific protein distribution,
756
Transmission, 33, 49, 159, 225, 231, 804.
AOBS, 57
contrast, 163–164
disk-scanning micro-lens array, 223–226,
227–229, 231, 235
dispersion, 683
of filters. See Filters
linear vs. log plots, 44–49
of glass fibers, 501–505
illuminator, 201, 127–128
losses due to refractive optics, 33, 217
table, 217
of objectives, 154, 158, 159–161, 641
relative, measurement, 26, 34, 36
table, transmission, 158, 159–161
of plant tissue, spectra, 416, 422
of Polaroid materials, 85
SHG signal detection, 707–709, 729–730
by small pinholes or slits, 225
Transmission electron microscope (TEM),
846.
correlated LM-TEM images, 852–855,
857–859
stereo images of platelets, 848–849
Transmission illuminator, ghost images,
201–202.
Transmission intensity, specimen thickness,
164.
Index
Transmittance, optical system, measured,
25–26.
table, 217
Transmitted light brightfield, 468, 472–473,
477.
blind deconvolution, 472–473, 477
Transparency, lighting models, 309–312.
Transverse electromagnetic modes (TEM)
laser, 83.
Trends, in laser design, 118.
Triple-dichroic, 33, 46, 48, 217–218, 678,
783.
light loss due to, 33
performance, 46–48
Triplet state, 103, 338, 339–342, 348,
362–363, 390, 516–518, 573, 646,
684, 691–693, 697, 698, 704, 852.
saturation, 339, 573
as a RESOLFT mechanism, 573
Triton X-100, 730, 852.
formaldehyde fixation, 370–372,
375–377
True color, 291.
TSM. See Tandem-scanning confocal
microscope.
TTL. See Transistor-transistor logic.
Tube length/chromatic corrections, table,
157.
Tunable lasers, 91, 103, 107, 109, 120.
broadband, table, 120
continuous wave dye, table, 91
diode, emerging techniques, 107
solid-state, 106, 109
solid-state ultrafast, 103
Tungsten carbide electrodes, radiance,
137–138.
Tungsten halogen source, 132, 137, 153.
Turnkey ultrafast laser systems, 118.
Tutorials, lasers by level, 124.
Tweezers, optical, 89–90, 110, 218, 383,
385.
setups for integration, 218
single-longitudinal-mode fiber laser for,
110
trapping wavelength, 89–90
Two-channel confocal images, 175–177,
177, 193, 425, 522.
A.thaliana, epidermal/mesophyll cells,
193, 425, 431–432, 434–436
Amoeba pseudopod, 169
colocalization, 667
display, 311, 841
FLIM, 522
harmonic images, 714–716
mouse muscles, 716
montaging, 331
neurons, 332
microglia, 396–398
eye, optic nerve, 481
Golghi-stained, 298
Lucifer-yellow, 314
rat-brain neurons, 398
transmitted light, 475
of peony petal, cytoplasmic, 175–176
rat intervertebral disk, 310–311
of zebrafish embryo, 177
Two-dimensional imaging, 60, 222,
397–398.
time lapse, 222, 397–398
Two-photon fluorescence excitation (2PE),
156, 160, 218, 535, 536, 750,
778–783.
chapter, 535–549
chromatic correction for, 156
for plant cells
advantages of, 778–779
cell viability, 779–781
vs. confocal microscopy, 779
dyes, 782
of green fluorescent protein, 782–783
pitfalls, 782
of thick specimens, 779
in vivo, 781
special objectives for, 160
visible and ultraviolet dyes, 218
Two-photon microscopy, 10–12, 195, 357,
535–549, 690, 697, 900–905. See
also, Multi-photon excitation; Multiphoton microscopy
autofluorescence, 545
basic principles, 535
of biofilms, 882–885
bleach planes, in fluorescent plastic, 193,
194
caged compounds, 544
calcium imaging, 545
chromophores, 543
2-photon absorption, 543
diagram, 540
detection, 538, 541
descanned, 542
non-descanned (whole area) detector,
541
stray light, 904
fluorescence, shadowing, 195
group delay dispersion, 5443
laser. 540–541
alignment, 900–904
monitoring, 901–903
mounting, 541
power level, 903–904
safety, 117–118, 839, 900,
903–904
living cell studies, review, 544–545
living animal studies, 545
minimize exposure during orientation,
905
mirror scanning, 543
optical aberrations, 542
photobleaching, 690, 697
practical tips, 900–905
beam alignment, 901
bleed-through, 904
choice of pulse length, 537, 903
pulse length, 109, 112, 115, 507, 537,
538, 902–903
983
specific specimens, see specimens by
name imaging multiple labels,
904–905
neurolucida protocol, 731
resolution, 539
and speed, 12
vs. spinning disk imaging. in plant cells,
783
stray light and non-descanned detection,
904
theory, 535, 537
wavelengths, 538–541, See also,
Botanical specimens
U
UBC 3D living-cell, microscopy course,
174, 183, 184, 190, 205, 364, 430,
435, 439, 805–806.
Ulbricht sphere, for measuring light, 140.
Ultrafast imaging, two dimensional, 222.
3D, 235
Ultrafast lasers, 88, 101, 103, 112–114.
Cr : Fosterite. 109, 114, 415, 541, 706,
707–709, 712–713
diode-pumped solid-state (DPSS), 112
distributed feedback (DFB) diode laser,
113
fiber, 113–114
table, 101
fiber-diode, mode-locked, 113
Nd : YAG, 88–89, 91, 95, 97, 103,
107–109, 111, 113–115, 117, 218,
245, 514, 680, 798
Nd : YLF, 89, 98, 100, 103, 109, 112–114,
750, 760–761
Nd : YVO4, 89, 95, 100, 103, 107–109,
111, 113–114, 541
solid-state, tunable, 103
spectrum, 44
titanium : sapphire, 112–113. See also,
Laser, titanium : sapphire; Titaniumsapphire laser
Ultrafast pulses, delivery by fiber optics, 88,
507.
dispersion losses, 502
Ultraviolet (UV), argon-ion laser lines, 85,
87, 90, 102, 339, 346.
other UV lasers, 111–117
use for micro-surgery, 218–219
Ultraviolet (UV) confocal microscopy, 109,
174, 195, 571.
absorption, 707, 713
autofluorescence, 431–432, 434, 544
CCD response, 29, 255, 459, 921–922
correct imaging with planapochromats,
14, 154
damage, 212, 290, 439, 544, 680, 686,
903
disk-scanners, 229
DNA-dyes, 782, 874. See also, DAPI;
Dyes GFP excitation, 798, 873
high-content screening, 811
ion-imaging, 346, 383, 529, 738, 742
984
Index
Ultraviolet (UV) confocal microscopy
(cont.)
multi-photon excitation, 535, 538, 544,
559, 646, 706, 905
photoactivation, 759
safety, 117–118, 839, 900, 903–904
simultaneous with DIC imaging, 846,
850
as source of stray signal in PMT
envelopes, 257
Ultraviolet performance of objective lenses,
154, 159–161, 706.
Ultraviolet widefield light sources, 132, 136,
139, 143, 226, 542.
table, 226
Ultraviolet transmission of optical fibers,
88.
Ultraviolet (UV) light, effects produced by
multiphoton intrapulse interference,
88.
Ultraviolet scanning light microscope, 6–7.
Uncaging, multi-photon microscopy, 383,
385, 545, 693, 760–764. See also,
Photoactivation.
Unconjugated bodipy/ceramide dyes, 760.
Under-sampling, 79, 635, 640, 652, 662,
831, 833, 836, 839, 841.
example, 640
uses, 68
Uniformity, of light source, 127–129.
Unit image body, 3D Airy figure, 147.
Upright vs. inverted microscope, 140, 157,
217, 230, 413, 722, 727, 870–872.
Unmixing. See Spectral unmixing;
structured illumination.
Up-conversion, fiber lasers, 110.
doped ZBLAN, 110
dual-ion doped, 110
UV. See Ultraviolet.
Video, 2, 4, 5–7, 11–14, 17, 37, 52–53,
61–62, 88, 219, 237, 261, 263, 346,
372, 430, 451, 505, 539, 554, 556,
589–590, 593, 604, 860, 885.
confocal, 25, 237, 914
impact on light microscopy, 5–7, 14
results, 14
signal, 258–259
Video-enhanced contrast microscopy,
imaging small features, 14, 68.
Vignetting, 210–211, 229, 245–247, 492,
541.
objective, off-axis performance, 245–247
Visibility, and signal-to-noise ratio, 37–38,
68. See also, Rose Criterion.
Visilog/Kheops, software, 282, 301–302,
312.
Visitech, confocal manufacturer,
descriptions, 88, 119–120, 226, 237,
908.
VT eye, 119–120, 908, 914
VT Infinity, 119–120, 908, 914
Visual cortex, identification of primary, 724.
Visual observation, magnification for, 146.
non-linearity, 72–73
Visualization, 280, 282–283. See also,
Multidimensional microscopy
images; Rendering.
definition, 280, 292
software packages for, table, 282–283
Vitrea2/Voxel View, software, 282, 335.
Volocity (software), 281, 236, 282, 295,
299, 312, 757, 762–764.
VolumeJ, software, 282, 304, 764.
VolVis, 281–282.
VoxBlast, 283, 301–302, 309, 312.
Voxel, defined, 20.
Voxel rendering, speed, 290.
Voxx, software, 283, 377, 764.
V
Vacuum avalanche photodiode (VAPD), 31,
254, 255.
definition, 254
schematic, 31, 255
VAPD. See Vacuum avalanche photodiode.
Vertical-cavity semiconductor diode laser
(VCSEL), 108.
Vibration.
compensation, 732
from cooling water, 84, 102, 499
of disk scanner, 753
causing distortion, 16, 39–41, 166, 201
of galvanometer mirrors, 40, 201
high-frequency, of acousto-optic devices,
55, 84
isolation, 85, 201, 219, 541
measurement, 30–41, 652
of mechanical shutters, 929
of objective lens motion, 754
optical fiber isolation, 505, 507
of optical fiber scrambler, 8, 84, 131
Vibronic laser, Ti : Sa four-level, 109.
W
WAD. See Whole-area; Non-descanned
detection.
Water, as immersion medium, 409, 410.
refractive index mismatch, table, 409, 410
two-edge response curves, 410
Water-coverslip interface, spherical
aberration generated at, 147.
Water-immersion objectives, 15, 23, 36,
141, 148–149, 154, 190, 235,
241–242, 247, 261, 377, 386–387,
389, 395, 411–412, 513, 542, 552,
556, 562, 567–568, 584, 654–656,
708, 727–728, 737, 747, 772. See
also, Spherical aberration.
4Pi, 562, 567–568
advantages, 149
biofilms, 870, 872
brain slices, 727–728, 730, 737
chapter, 404–413
correction-color/flatness/transmission,
154
deep imaging, 395
dipping objectives, 161, 209, 411, 429,
568, 613, 727, 737, 870, 872
in fluorescence ion measurement, 737
ion measurement, 737
living cells, 386–387, 389, 395, 398
performance measured, 47, 655–656
plant cells, 429, 433, 772
STED, 576
transmission curves, 159–161
use and limitations, 15
Watershed algorithm, 322–325, 777, 822.
for segmentation, plant cell images, 777
Wave optics, 4, 10.
for calculating axial resolution, 4, 146,
154
Wavefront error, 217.
lower, with hard coatings on filters, 45
Wavelength, 24, 28, 43–51, 62, 88, 107,
114–115, 118, 129–130, 135–139,
165–166.
calculation of Forster radius, FRET, 793
and CCD coupling tube magnification, 62
filters for selecting, 43, 44, 88
in multi-photon lasers, 165–166. 415, 750
multiple, dynamic embryo analysis, 756
of non-laser light sources, 129–130,
135–136
and optimal zoom setting, 24
vs. pinhole size, 28
selecting, with interference filter, 88,
165–166
stability, in non-laser light sources,
137–139
tunability, of lasers, 107, 109.
Wavelength expansion, non-linear, 114–115.
Wavelength ratioing, 346. See also, FRET;
FLIM.
Wavelength response, chromatic aberration,
663.
Wavelength-selective filters, 43–51, 88.
Wavelength-tunable lasers, summary, 107,
113, 116, 118, 550.
Wavelet compression, 581–584.
Wavelet de-noising protocol, 733–734,
819–820.
Waxes, plant, 420, 428, 434–435, 714–715.
Website references, 123.
2 photon excitation spectra, 546, 727,
729, 782
brain slices, 727
CCDs, 76, 234, 927, 931
components, 58
confocal Listserve, 390, 901
deconvolution, 495
dyes, 221, 343–344, 782
fluorescent beads, 653
FRET technique, 185, 803
high-content screening systems, 811
image management, 865
lasers, 104, 115, 120, 123–125
live-cell chambers, 388–389, 870
movies related to book, 235, 392
muscles, 237
Index
non-laser light sources, 138, 143
plants, 769
safety, 117–118, 839, 900, 903–904
software, 282, 376, 594, 734, 762, 764,
776, 777, 820, 824, 827, 831–833,
844, 845, 864–862, 865–867, 869
SPIM, 672
Wedge, compensator, 566–567.
Wedge, rotating, for light scrambling, 84,
131.
Wedge error, in interference filters, 45–46,
151, 211–212, 630.
in traditional filters, 45
Wedged fiber-optics, reduce reflections, 85.
Well-by-well data, 817.
WF. See Widefield.
WFF. See Widefield fluorescence
microscopy.
White light continuum lasers, 88, 109, 113
continuum, 88, 109
He : Cd, 113.
Whole-area and external detection, 541–542.
See also, Non-descanned detectors.
Whole-cell patch pipet delivery, 360,
726–727.
Widefield deconvolution, 751–753, 785. See
also, Deconvolution.
botanical specimens, 785
for living imaging, 751–753
Widefield (WF) fluorescence microscopy, 3,
22–23, 26, 172–173, 219, 453–467,
518. See also, Epifluorescence
microscopy, Deconvolution.
compared to confocal, 453–467, 644–647
CCD/confocal comparison, 458–459,
465
same specimen, 465, 482
compared to structured illumination, 274
deconvolution, imaging living cells, 23,
392
deconvolving confocal data, 461–464,
466
fluorescence detection, 459–460
fluorescence excitation, 459
fluorescence lifetime imaging, 518
gain-register CCDs, 460–461
images utilizing out-of-focus light, 26
imaging as convolution, 453–457
imaging thin specimens, 172–173
integration of fluorescence intensity,
459
interaction of photons with specimen,
22–23
light-emitting diode sources, 136
limits, linearity/shift-invariance, 457, 490,
564
model specimens, 461
noise, 459–463
optical sectioning schematic, 469
optical tweezers/cutters, 219, 89, 383, 385
out-of-focus light, 461
point-spread function, 453–457, 459–463
resolution, 3
sensitivity, 459–463
single point images, 454
pros/cons, 644–648
table, 459
temporal resolution, 458
Wiener filtering, 494, 496. See also,
Gaussian filtering.
image enhancement, 496
image restoration by, image, 494
Windows software, for automated confocal,
810.
WinZip, 580.
Wollaston prisms, DIC, 156, 468, 473, 475.
See also, Nomarski; DIC contrast.
Working distance (WD) of objective lenses,
5, 9, 129, 145, 154, 157, 198, 249,
511, 568, 598, 634, 673, 678,
727–728, 747, 774, 779, 781, 872.
table, 157–158
WORM disks (write once, read many), 586.
X
Xenon arc lamps, 44, 132, 137–138, 144.
iso-intensity plot of discharge, 132
pulsed-operation, 137–138
shapes of electrodes, 132
spectral distribution, 144
super-pressure, spectrum, 44, 136
explosion hazard, 136
wavelengths available for detection, 44
Xenon/iodine fill arc, radiance, 137–138.
Xenopus laevis, 13, 610, 746, 748–753.
blastomere, 757
confocal/multi-photon comparison, 750
embryo
viewed with confocal, 748–753
viewed with OCT, 610, 749
embryo viewed with MRM, 623–264
in situ imaging, 746, 748
oocyte wound closure, 749
X-Y resolution, confocal/widefield
compared, 36.
Y
Yellow fluorescent protein (YFP), 221–222,
429.
FRET pair with CFP, 791–803
YFP, 221–222, 429
Yokogawa disk-scanning confocal system, 6,
12–13, 16, 216, 224–226, 231,
234–237, 458, 754.
CSU-10/22 model, 223, 231, 236, 915
with EM-CCD, 234, 237, 755
high speed acquisition, 11, 220, 222–226,
229, 231, 458, 667, 754, 784
results, 236–237, 755, 783
vibration, 16
Ytterbium tungstate (Yb : KGW) laser, 108.
Z
ZBLAN up-conversion glass fiber, 110.
Z-buffering, 304–305.
Z-contrast, in confocal microscopy, 180.
985
Zea mays. See Maize.
Zebrafish, 174, 176, 761.
GFP image, 176, 176
autofluorescence, 174
pancreas expressing DsRed, 176
scatter labeling/lineage tracers, 761
Zeiss, confocal manufacturer, 212, 214,
217, 226, 231–232, 655, 771,
916–917.
510 META confocal microscope, 655,
908, 916
Achrogate beam-splitter/LSM 5-Live, 50,
119–120, 212, 231–232, 916
Axioimager system, 217
fluorescence correlation spectrometer
(FCS), 383, 385, 602, 801, 803, 805,
917
HBO-100 source, self-aligning, 134–135
high-content screening, 811
LSM 5-Live line-scanning confocal
microscope, 50, 51, 231–232, 237,
784, 908, 916
META confocal spectral detector, 51,
119–120, 161, 202, 660, 663, 796,
916.
mini-PMT arrays, 51, 667FRET, 706
tests, 663
objectives, advantages of, 155–156
Infinity Color-corrected System, 155,
217
plan objectives, table, 152
transmission specifications, 161
tube length conventions, 157, 239
working distance of objectives, table,
158
Zernike moments, 247–249, 818–820.
Zernike polynomial fit, 245–247.
table, 247
wavefront aberration function, 247
Zinc selenide (ZnSe) diode lasers, 106.
Zirconium arc lamps, 136, 141.
spectrum, 136
Zone System (Ansel Adams), 71–72.
Zoom magnification, 11, 24, 37, 63–64, 66,
70. See also Magnification
optimal, 24
optical vs. electronic bandwidths, 70
relationship to area scanned, 63
Z-position and pinhole/slit size, 227.
Z-resolution, 3–4, 22, 36, 149–150, 224,
225–228, 563, 752. See also, Axial
resolution.
4Pi microscopy, 563
in confocal fluorescence microscopy, 36
effect, of fluorescence saturation, 22
improvement, 752
of pinhole disks, 224
in STED, 576
Z-scanners, evaluating, 215.
Z-sectioning, imaging brain slices, 729.
Z-stack, 23, 754.
of images of cheek-cell specimen, 23
speed acquisition constraint, 754