Download Basics of Light Microscopy

Week-long microscopy course
Class 2
http://microscopy.duke.edu/
[email protected]
The problem with standard microscopes
and biology
Sectioning
Optical sectioning of thick samples
3D reconstruction
The confocal
principle
How a laser scanning confocal microscope
works
http://www.olympusfluoview.com/theory/index.html
What a confocal looks like
Scanhead
A microscope
(inverted or upright)
Lasers
and
electronics
Probably on an air table
Computer
Confocals have lots of adjustability
LASERs are used for excitation
Ideal for point scanning:
•Narrow collimated beam, low divergence
•Powerful
http://www.olympusfluoview.com/theory/laserintro.html
Many lasers available . . .
Gas lasers
Diode lasers
(Kr/Ar
488 568 647)
405
Ar/Ar
458 488
488
HeNe
543
594
633
514
561
635
(Color coding refers to the color of the fluorophore
for which the laser line is most commonly used)
Adjusting the laser power
Relatively fixed output
Rapid control 0-100% T
Laser
AOTF
Laser
AOTF
l selection and intensity control
Fluorophore saturation
Bleaching is proportionately
worse here
Emission
intensity
Image will suffer loss of contrast and
quantification issues
Excitation intensity
Widefield is normally in the linear range, the concentrated
laser spot in confocal may not be.
Start low and increase to the minimum necessary
Photomultiplier tubes (PMTs) are used as
detectors
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•
•
•
•
•
Fast (good for scanning)
Large collection area
Good SNR
Very large dynamic range (with gain&offset adjustment)
Adequate dynamic range at a single gain&offset position
QE <30% (not as good as CCD)
Gain and offset adjustments
100 msec
200 msec
400 msec
600 msec
800 msec
1000 msec
2000 msec
Camera:
Confocal:
Increasing gain (voltage on PMT)
Offset to set the background to black
Optimal gain and offset
Confocals have special display modes to highlight saturated
and 0 intensity pixels
Should you have no, a few or many saturated and zero intensity pixels?
Scanning mirrors
The relative position of the two mirrors can point the spot
anywhere in the field
http://www.olympusconfocal.com/theory/confocalscanningsystems.html
Scan speed
Fast scans
• Fast processes
• Useful for focusing and
adjustments – eg 1 fps
Slower scans
• More light gathered
• Better images
• More damage
Averaging
Scanning each pixel multiple times and averaging
improves the noisy signal . . .
Averaging: how much do you need?
1
2
8
4
16
Decreasing rate of improvement, empirically determine a good balance
between final SNR and time/damage in acquisition
Quiz B
Scan area: zoom
The area swept by the galvo mirrors can be adjusted . . .
Is this meaningful zoom or just digital zoom? . . .
Scan area and number of pixels
Any particular frame can have different numbers of
pixels. . .
How do I set these two things? How many pixels
do I need in my image for the best resolution?
Same area:
225 mm across
225 mm/512
= 440 nm
225 mm/1024
= 220 nm
The Leica SP5 goes up to 8K by 8K, shall we have 64 Mpx for
every scan?
What was a pixel? Part 1.
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Shall we have as many px as possible?
How big should a pixel be?
Are trade-offs involved?
Are pixels real?
What is a grey value?
How many pixels do I need in my image for the
best resolution?
Nyquist sampling theorem: Sample at twice the resolution
Resolution
=
0.61 l / NA
Increasing number of pixels per area
Signal
under-sampled
Not capturing all
the resolution of
the system
Signal
Well sampled
Just right,
The Nyquist rate
Signal
Over-sampled
Not gaining any
more resolution,
more bleaching
waste of time and
disk space
Do I really need to listen to Nyquist?
• You might not always be seeking the best resolution
• You might need to under-sample for speed,
phototoxicity . . .
• Over-sampling is effectively averaging
But optimal sampling is important and beneficial
in many cases
Lateral resolution in confocal: Theory
At best, the lateral resolution of a confocal is better
than widefield by a factor of √2.
wf
confocal
Essentially the effective airy
disk is the product of
illumination and detection
disks so has steeper sides
Lateral resolution in confocal: Reality
But to see this benefit the pinhole needs to be at <0.25
AU.
rxy
Relative
intensity
0.61 l
√2 NA
10%
~ 0.61 l
NA
~ 0.61 l
NA
100%
Up to 1000%
Quiz C
Multi-channel confocals
Most confocals have several
laser lines
and more than one PMT
Why are fluorescence scopes generally
not built like this?
Simultaneous or sequential acquisition
Blue
Green
Red
Faster
Blue then Green then Red
Less bleedthrough
Bleedthrough
DAPI
AF488
• Down the spectrum (1st law of thermodynamics)
• Worse when intensities are unbalanced
Line vs frame switching
Blue line1 then Green line1 then Red line1
Blue line2 then Green line2 then Red line2
The AOTFs switch the laser light
on and off very rapidly
Good to see all the channels
appearing together
Blue image then
Green image then
Red image
Good when something physical
happens between colours (eg
change dichroic)
Use this when you have more
channels than PMTs
3D acquisition
Confocals are good for acquiring stacks of images because
of the optical sectioning ability
Lateral and axial resolution
XY
rxy ~ 0.61 l / NA
YZ
rz  l / NA2
Resolution is always worse in Z than XY
Optical section thickness
10x/0.3
20x/0.45
Optical section (micron)
Smaller with
higher NA
63x/1.2
40x/1.3
63x/1.4 100x/1.4
Larger with
more open PH
Pinhole size (AU)
Sampling in the z-axis
The same principle as in XY
Some regions not
imaged
Covered
Covered and well
sampled
Imaging big things
Macroscope | Stereoscope
Macro/stereoscope imaging modes
Stitching/tiling/montage/mosaic
Two photon excitation
Single photon
Two-photon
Excitation is limited to a
small focal volume where
photons are most
concentrated
Differences to a single photon confocal
Can use all the light,
no pinhole needed
2-photon advantages
Main advantage: Imaging thicker specimens
The longer wavelength excitation penetrates further into the sample
(less scatter and absorption in IR). The scattered excitation light
doesn’t cause background fluorescence.
The excitation is also not attenuated by fluorophore absorption above
the plane of focus
Emission advantage
Because we are able to image all the light (no pinhole)
this is less affected by scatter
With pinhole
No pinhole
The NDD is closer and more efficient for a scattered
beam path (which is hard to move efficiently through
several lenses)
1P
2P
Pretty pictures from the Olympus FV1000 MPE brochure
A Pulsed laser is required for MPE
For efficient MPE we need photon
concentration in space and time . . .
http://www.olympusmicro.com/primer/techniques/fluorescence/multiphoton/multiphotonintro.html
A pulsed laser
Chameleon Ultra II
Femtosecond pulsed laser
3-4 W of power at peak
Tunable 680-1080 nm @40 nm/sec
This gives flexibility, since these laser cost about $200,000 each
its not normally practical to have several per machine like with 1 photon
Fluorophores for 2-photon
Some fluorophores are
pretty different
between 1P and 2P
Excitation spectra not
exactly double 1photon (tends to be
broader and blue
shifted)
Quantum dots can be
excited at nearly any l
Fluorescent proteins
are pretty good
Photobleaching and toxicity
Photodamage in 2P is confined
to the thin layer being imaged
Power
(photons/mm2
Wide-field
X
1 Photon
105X
2 Photon
(average)
106X
2 Photon (peak)
1011X
The bleaching in that small area
is probably worse than 1P (some
photodamage is highly nonlinear)
In general, for thick samples 2P has an advantage over 1P
For thin samples, 2P is often worse than 1P
SCALE reagent
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•
•
•
4 M urea
10% (wt/vol) Glycerol
0.1% (wt/vol) Triton X-100
pH of 7.7
Refractive index of 1.387 and 486 nm
Soak your sample in it for a couple of weeks . . .
http://www.nature.com/neuro/journal/v14/n11/full/nn.2928.html
It clears fixed samples by
removing refractive index
changes
Doesn’t destroy fluorescence
Preserves tissue structure
And allows . . .
2-photon vs. 1-photon
•Improved SNR in deep-tissue imaging (especially with NDDs)
•The IR 2PE is less phototoxic in many cases, especially for UV
dyes.
•Photobleaching/damage restricted to plane being imaged.
Photobleaching or uncaging is possible with fine z-axis resolution
• XY resolution is slightly less good
•Multi-channel acquisition is harder (excitation cross-sections are
normally much broader) and limited in excitation l
•No control of optical section thickness
• Lasers are expensive and require exact alignment and can
produce heating and other damage
Don’t use a 2-photon system unless you need the advantages
Fast confocals
Other ways of doing something like this
Without these problems . . .
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What can we do about this?
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The spinning disk principle
The spinning disk principle
Detector comparison
Photodetectors
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
Photobleaching advantages
t
t
m
How does a spinning disk work?
Sectioning in spinning disks
Ideal pinhole diameter
= 0.5 l M/NA
• 100x/1.4 = 20 mm
• 20x/0.5 = 11 mm
What works well on a spinning disk
Living things that need sectioning . . .
• Things which match the high magnification, high NA
optimizations (eg subcellular imaging)
• Photosensitive samples
• Fast imaging
Quiz D
Deconvolution
A mathematical post-acquisition processing of images to reduce
the blur from out of focus light. This can increase the signal to
noise ratio and resolution of the image.
The Point-Spread Function and image
formation
XZ
XY
PSF
Image
The image is the sum of all blurred point images
Convolution
An image is a convolution of the object:
Object
x
PSF
=
Image
XY
XZ
The aim of De-Convolution
Image(s)
Knowledge of
imaging psf
Underlying
object
The PSF can be measured
or predicted
Two types of deconvolution
1. Deblurring/nearest neighbor/2D deconvolution
– Estimates the blur from other focal planes and removes it
– Sharpens the image but is non-quantitative
– Very fast, real time
XZ
Blur the image from here
and remove it from the
images above and below
Two types of deconvolution
2. Restoration/3D deconvolution
o Iterative reassignment of photons based on modeled
convolution
o Works in 3D (ie considers all the data together)
o More computational intensive, takes a few seconds to minutes
o Conservative and quantitative
o Better able to cope with low SNRs
Move this photon
from the blur
to the object
Deblurring v restoration
A Workingperson’s Guide to Deconvolution in Light Microscopy
The computational process
Original
image
Estimated
Object
psf
convolve
Predicted
Image
Constraints
n iterations
The psf is improved
in blind
deconvolution
Photon
reassignment
Compare to
original image
Stopping criteria:
If the same, the
object perfectly
accounts for the
image
Finished
What actually improves during deconvolution?
• Photon reassignment:
• Noise
Blur
Structure
. . . So higher SNR
Which modalities does it work with?
All of them but the relative benefits are different for the
different modalities
Widefield
PSF calculated for
60x/1.4 NA
objective
Green fluor
= 2 mm
Spinning
disk
Confocal
(1 AU)
Limited in widefield for thick samples
Too many
PSFs to draw
Image:
Sharp image
+dimmer blur
Overwhelmed by blur
Summary: What deconvolution is/ is not
Post-processing that works for all (3D) imaging modalities
Fairly computational intensive to calculate properly
It needs good images - Does NOT allow you to take awful
images and magically transform them!
Good for live cell imaging: gentle but slightly noisy images +
deconvolution = good images with less phototoxicity
It doesn’t replace confocal. Anything >30 mm and the blur
becomes too much for processing and you need a spatial
filter.
TIRF
Total Internal Reflection
Some reflected, some refracted
Evanescent wave
Evanescent wave
Exponential decay
of intensity
Iz = I0e-bz
Evanescent wave
Evanescent wave
Plane of excitation
~100 nm thick
Two ways of generating and imaging TIRF
Prism-based
A bit more to align
Better SNR, lower background
Slight constraint on imaging objective
Sample access is difficult in some setups
Have to build your own
Objective-based
More convenient
Needs >1.45 NA objective
SNR still very good
Components of a TIRF system
TIRF angle
autoalignment
optics round
the back
Opaque
incubator
Fiber-coupled
lasers
EMCCD
Inverted microscope with a special TIRF objective
Widefield vs TIRF
Myosin Actin
What is TIRF good for?
Anything at the edge of the cell/tissue
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Exocytosis/endocytosis
Vesicle dynamics
Cytoskeletal activity at the membrane – Focal adhesions
Signalling in the membrane – translocation
Relatively distinct
subset of samples gain
from TIRF imaging . . .
Tools available for you
Choose wisely
Further information
Review articles about confocal theory
http://www.olympusfluoview.com/theory/index.html
Java tutorials to play with
http://www.olympusfluoview.com/java/index.html
SVI wiki on 3D Microscopy and deconvolution
http://support.svi.nl/wiki/
Deconvolution review articles
http://zeiss-campus.magnet.fsu.edu/referencelibrary/deconvolution.html
Multiphoton articles/tutorials
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonhome.html
Reviews on multiphoton imaging
http://zeiss-campus.magnet.fsu.edu/referencelibrary/multiphoton.html
TIRF
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html
SPIM
http://dev.biologists.org/content/136/12/1963
Spinning disks
http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html