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College of Medicine, Biological Sciences and Psychology
Core Biotechnology Services (CBS)
Advanced microscopy and bio-imaging I
Dr Kees Straatman
Manager of Advanced Imaging Facilities (AIF)
10 -12 am:
Introduction
Fluorescence microscopy
Advanced imaging systems
2 – 4 pm :
Fluorescence imaging
Advanced fluorescence techniques
Facility
April 2013
Renaissance in biological imaging
 Genomics, proteomics, metabolomics
 Anti-body development
 Introduction of more stable fluorochromes
 Introduction of fluorescent proteins
 Introduction of more sensitive/faster detectors
 Introduction of new imaging platforms
 Advances in computer development/data processing
April 2013
Renaissance in biological microscopy
Whole animal imaging systems
(Maestro, CRi)
R. Weissleder, U. Mahmood and J. Tam,
Massachusetts General Hospital
Super resolution microscopy;
STORM images of mammalian
mitochondria.
Zhuang Research Group, Department of Chemistry and
Chemical Biology, Harvard University, Cambridge, MA
April 2013
Renaissance in biological imaging
 Combination of existing and new technologies
IVIS Spectrum Bioluminescence and Quantum MicroCT scanners
April 2013
Renaissance in biological imaging
 Combination of existing and new technologies
Correlative Light and Electron Microscopy (CLEM)
Daguenet et al. (2012) Mol Biol Cell 23: 1765-1782
April 2013
Simple microscope
Hans Loncke
Two Trinacria
species of
about 0,1
mm size
Van Leeuwenhoek’s microscope
Around 1668
July 2007 Micscape Magazine
April 2013
Compound microscope
Microscope built by Zacharias Janssen,
probably with the help of his father, in
the year 1595. Considered the first
microscope.
Robert Hooke.
First to use the word ‘cell’ while
looking at a piece of cork.
First publication with drawing of a
microorganism (microfungus Mucor)
April 2013
Compound microscope
CO2 and temperature control
Incubator
Joystick to
move stage
Light source (laser)
Camera
Antivibration
table
April 2013
Aberrations
Around 1668
Up to 275x mag
1595
3-9x magnification
April 2013
Aberrations
Objective lens
Chromatic correction
Plan Achromat
Blue (486 nm) and red (656 nm)
Plan Fluorite
blue, red and green (588 nm)
Plan Apochromats
Also corrected for 436 nm
SuperApochromats (SAPO; Olympus)
CFI Plan Apochromat VC - Nikon
Infinity Colour-Corrected - Zeiss
U-V-I – Leica
Corrected from UV to the near
infrared region
Plan: flat-field /spherical aberration corrections
April 2013
Aberrations
April 2013
Aberrations
Coverslip correction
April 2013
Aberrations
Coverslip correction
Coverslip
number
Coverslip
thickness (mm)
#0
0.08 - 0.12
#1
0.13 - 0.17
#1.5
0.16 - 0.19
#2
0.17 - 0.25
#3
0.25 – 0.35
#4
0.43 – 0.64
Calculated intensities using a dry
objective
April 2013
Image Quality
The quality of a microscope image is assessed by the
following:
Focus - Is the image blurry or well-defined?
Resolution
 Spatial resolution: the ability to visualize two points as
separate points
 Temporal resolution: frequency at which images are
recorded/captured
April 2013
Resolution
1872: Ernst Abbe formulates his ‘wave theory of microscopic imaging’ :
d = λ 
2η sin
η = refractive index of medium
η.sin = NA (numerical aperture)
diffraction limited microscope
April 2013
Resolution
1872: Ernst Abbe formulates his ‘wave theory of microscopic imaging’ :
d = λ 
2η sin
η = refractive index of medium
η.sin = NA (numerical aperture)
d = 0.5 * λ
NA
Refractive index (η ): the light-bending ability of a
medium.
April 2013
Resolution
April 2013
Resolution
We want to resolve 2 points!
The best focused spot of light that a
perfect lens with a circular aperture
can make, is limited by the
diffraction of light.
Diffraction limited microscope
R = 0.61λ /NA (Rayleigh criterion)
d = 0.5 λ/NA (Abbe)
http://micro.magnet.fsu.edu/primer/java/imageformation/rayleighdisks/index.html
April 2013
Resolution
• Radial resolution:
d = 0.5λ /NA
R = 0.61λ /NA
400 nm
488 nm
633 nm
300
250
200
633
150
488
100
400
50
0
15
36
57
78
99
120
141
162
183
204
225
246
267
288
309
330
Excitation:
April 2013
Resolution
April 2013
Resolution
Axial resolution:
XY
YZ
d = 2λη/(NA)2
λ
XY (nm)
Z (nm)
488
231
754
561
244
867
XY (2D): pixel
XYZ (2D): voxel
(NA = 1.4; η = 1.515)
XZ
April 2013
Objectives
d = 0.5 λ /NA
R = 0.61 λ /NA
But magnification has influence on optimal pixel size of CCD camera. Size of
field of view for a fixed CCD chip.
April 2013
Köhler illumination
To obtain optimum contrast and resolution in brightfield microscope
•Focus your sample
•Close the field diaphragm
•Focus the condenser
Adapted from: http://biology.fullerton.edu/facilities/em/BrightSetup.html
April 2013
Köhler illumination
To obtain optimum contrast and resolution in brightfield microscope
•Focus your sample
•Close the field diaphragm
•Focus the condenser
•Centre the condenser
•Open field diaphragm till
whole view is filled
Adapted from: http://biology.fullerton.edu/facilities/em/BrightSetup.html
April 2013
Contrast
No colour and too little contrast between structures with similar
transparency.
Solution 1: phase contrast microscope: first described in 1934 by
Dutch physicist Frits Zernike; Nobel Prize for Physics, 1953 .
It translate minute variations in phase into corresponding changes in
amplitude, which can be visualized as differences in image contrast.
April 2013
Phase contrast
April 2013
Contrast
No colour and too little contrast between structures with similar
transparency.
Solution 2: Differential interference contrast (DIC) microscopy
uses polarized light with specialized beamsplitting (modified
Wollaston or Nomarski) prisms.
April 2013
DIC
Only single cell or thin layer of cells are
observable.
Two other options:
Darkfield microscopy
Polarization microscopy
April 2013
Contrast
No colour and too little contrast between structures with similar
transparency.
Solution 3: stain your
sample with colour dyes.
Solution 4: Fluorescence
microscopy
Blue = DNA;
Green = talin;
Red = actin;
Hematoxylin stain (histology)
April 2013
Objectives
April 2013
Objective colour codes
April 2013
Fluorescence microscopy
 Advantages
–
–
–
–
Very sensitive (can detect single molecules)
Can be used in vivo
Localization of proteins
Good time resolution
Disadvantages
 Usually requires a fluorescent label
 Excitation light can be damaging
(phototoxicity, bleaching)
 Often time consuming
 Quantitative imaging is challenging
April 2013
Luminescence
Excitation of a molecule resulting in emission of light.
Chemoluminescence: resulting of a chemical reaction
 Bioluminescence: by a living organism
April 2013
Bioluminescence
Best know is the firefly luciferase :
Firefly
Luciferase (reporter gene)
luciferin + ATP + O2
Oxyluciferin + AMP + PPi + CO2 + light
Emission peak ~ 560 nm
(PPi = pyrophosphate)
Renilla
luciferase
Coelenterazine + O2
Coelenteramide + CO2 + light
Emission peak ~ 480 nm
April 2013
Bioluminescence
The green-emitting
luciferase was derived
from a Japanese
luminous beetle
(λmax 560 nm); the redemitting luciferase was
derived from railroad
worm (λmax =
630 nm)
Kwon et al. (2010) BioTechniques 48: 460-462
April 2013
Luminescence
Excitation of a molecule resulting in emission of light.
Chemoluminescence: resulting of a chemical reaction
 Bioluminescence: by a living organism
Photoluminescence: absorption of photons causing re-radiation
of photons
 Phosphorescence: delayed radiation
 Fluorescence: instant radiation
April 2013
Fluorescence
Jablonski diagram
Absorption of light with a short λ
resulting in emission of light with a
longer λ (The so-called Stokes shift)
Fluorochrome: molecule that is fluorescent.
Fluorophore: a component of a molecule which causes this molecule to be fluorescent.
April 2013
Fluorescence microscopes
April 2013
Filters
The Stokes shift we can use to separate the excitation and emission light in
fluorescence microscope
41
Filters
• Exciter D470/40x
• Dichroic 495DCLP
• Emitter E515LPv2
Old filter
April 2013
Filters
• Exciter ET470/40x
• Dichroic T495LP
• Emitter ET525/50m
New filter
April 2013
Filters
• Exciter s D350/50x;
• Dichroic 62002BS
• Emitter 61002m
• S492/18x;
•
S572/23x
April 2013
Filters
Filter wheel
April 2013
Filters
Prism based
Borlinghaus and Kuschel; Nature Methods - 3, (2006)
diffraction grating
April 2013
Light sources
 Arc bulb
Mercury Arc lamps (e.g. X-cite)
Xenon Arc lamps
Metal Halide Arc lamps
 LED (light-emitting diode )
 Monochromator
 Laser
April 2013
Light sources
 Arc bulb


Need long time to warm up
Flickering, increases over time. Associated with
inadequate cooling of the lamp. Most unstable light
source in use for microscopy these days
April 2013
Light sources
 Arc bulb
Xenon is relatively weak but has a more continuous and uniform
spectrum what makes it more preferable for quantitative imaging
April 2013
Light sources
 LED
• No warming-up/cooling-down
• Fast switching
• Low power consumption
• High emission stability (does not change with brightness)
• Extremely long life span
• Minimal heat output
• Compact size
April 2013
Light sources
 Monochromator
April 2013
Light sources
 Laser
• Single wavelength
• High power
• White laser (supercontinuum laser)
• A laser can be focused or non-focussed
April 2013
Detectors
 CCD (charge-coupled device)
 EM-CCD (electron-multiplying CCD)
 CMOS (complementary-metal-oxide-semiconductor
detector); much higher readout speed.
 PMT (photomultiplier tube)
April 2013
Detectors
3 types of noise systems:
 Dark current noise – noise from heat and cosmic noise exposure dependent. Reduce by cooling camera.
 Read noise – noise of reading the signal - fixed; not PMTs
 Photon shot noise – square route of signal - signal
dependent
Signal to noise (S/N or SNR) is a measure for the quality of the system
All values must be compared in electrons
SNR = QE*S / V(NoiseDark) 2 + (NoiseRead)2 + (QE*S)
S = Signal in Photons
QE = Quantum efficiency
Online Signal to Noise calculators exist; see e.g. www.photomet.com
April 2013
Detectors
• PMT (image pixel by pixel)
A small fraction of the collected photons (less than 30%) generate
photoelectrons which get amplified by a factor of about 1 million;
Depending on the voltage.
Dark noise; single scan with standardized gain and offset without sample
Photon shot noise; square root of signal
SNR = S/V(NoiseDark)2 + (NoiseShot)2
Alternative: SNR = (Signal – Background)/Standard Deviation of Background
April 2013
Detectors
CCD read out
CMOS reads out pixel by pixel
 Fast
 Noise/pixel
EM-CCD camera has an
Amplification stap before
output node
 More sensitive
 Extra noise (excess noise
factor)
Output node
Read noise (fixed)
April 2013
Detectors
Cooled to -70 to -90 °C
Reduced dark current
Coates et al (2009) Scientific CMOS Technology A High-Performance
Imaging Breakthrough White Paper
April 2013
Detectors
Photometrics
Noise limited image i.e. the signals
below the noise cannot be seen
EMCCD: minimizing the
read noise and dark current
April 2013
Detectors
EM-CCDs Hamamatsu
April 2013
CCD Spatial Resolution
Image
Pixels
Digital Image
To meet resolution of the microscope,
pixel size should be at least 1/2 airy disk size; ideally 1/3.
Known as The Nyquist criterion: the minimal sampling density needed to capture ALL
information from the microscope into the image.
However, smaller pixels collect less photons, have less full well capacity and results in
slower read outs of the chip.
April 2013
Optimal resolution CCD
Objective NA
Projected size
on CCD (µm2)
(R * Mag.)
Optimal pixel
size (µm2)
40x 1.3
10.4
3.5 – 5.2
60x 1.4
14.4
4.8 – 7.2
100x 1.4
24.0
8 - 12
R = 0.61λ /NA with λ = 550nm
Camera pixel size = 6.45x6.45 µm. So without much loss of resolution you could
image with a 2x2 binning using the 100x objective
April 2013
Binning
Binning increases image intensity
while decreasing resolution & transfer time
April 2013
Binning
50ms, no binning
Intensity max 746
Resolution 240nm
50ms, 2x2 bin
Intensity max 2297
Resolution 480 nm
Improved S/N
Images collected by JWS in the Nikon Imaging Center at Harvard Medical School
April 2013
Image Quality
The quality of a microscope image is assessed by the
following:
Noise – S/N
Focus - Is the image blurry or well-defined?
Resolution – What is the minimal distance between two points in
the image that still can be seen as two separate points?
Brightness - How light or dark is the image?
Contrast - What is the difference in colour and light between
adjacent areas of the specimen?
April 2013
Bit Depth vs. Dynamic Range
 Bit Depth is determined by how many electrons are used to equal one gray level.
 As bit depth increases, the same original information is divided into ever smaller
increments.
Bit depth
Greyscale
levels
1
2
2
4
4
16
8
256
12
4,096
16
65,384
April 2013
Exposure Time
687
1051
1858
3260
3888
Image maximum grayscale value
12-bit camera maximum = 4095
Increasing exposure time increases signal
Images collected by JWS in the Nikon Imaging Center at Harvard Medical School
April 2013
Gain (CCD)
Increasing gain reduces the number of photons / gray scale value
Increasing gain, same exposure time
Images collected by JWS in the Nikon Imaging Center at Harvard Medical School
April 2013
Advanced imaging systems
Haze and blur
• In a normal epi-fluorescence microscope
you collect light from the focal plane and
light from below and above the focal plane.
Epi-fluorescence
Perfect focus
April 2013
Epi-fluorescence microscope
Live cell imaging
Multiple colours
Time series
Multiple position
Multiple z-sections
April 2013
Deconvolution microscope
Widefield PSF
SVI
Using maths to remove haze and blur
April 2013
Deconvolution microscope
192 optical sections of a fruit fly
embryo leg acquired in 0.4-micrometer
z-axis steps with a widefield
fluorescence microscope
April 2013
Confocal laser scanning microscope
sample
3D-reconstruction
Z-series
First confocals in the 50s
First commercial system in 1987
3D pixel = voxel
April 2013
Confocal laser scanning microscope
Optical sections
3D-reconstruction
Blue = DNA
Green = Centrosome
Red = Microtubules
April 2013
April 2013
Resolution CLSM
128x128
256x256
512x512
1024x1024
April 2013
Confocal laser scanning microscope
Deconvolution
Set top
Set bottom
April 2013
Spinning disk microscope
CLSM
30° turn is 1 image; 360° is 12 images ~ 2000 images/second.
However, exposure time might be 100 milliseconds
(quite common when using fluorescent proteins in living cells).
April 2013
Spinning disk microscope
widefield
Spinning disk
CLSM
Z
XY
PSF
Stefan Terjung, EMBL
April 2013
Multi-photon laser scanning microscope
April 2013
Multi-photon laser scanning microscope
April 2013
Multi-photon laser scanning microscope
Even scattered fluorescence photons are usefull in 2photon regime
April 2013
Multi-photon laser scanning microscope
• Penetration depth CLSM ~ 30-50 µm
 Use of pulsed infrared laser increases penetration
depth to up to 1000 µm
 Used for imaging of thicker samples like mouse
embryos, mammalian brain, intact nervous
system, bone-marrow.
April 2013
Multi-photon laser scanning microscope
CLSM (50 µm)
Mouse cortical
pyramidal neurons
expressing EYFP.
Excitation using 920 nm.
(Zeiss LSM 7 MP
brochure)
Transgenic mice expressing a mixture of
different fluorescence proteins but individual
neurons expres each a unique combination
of this mixture (brainbow).
Williams et al. (2010) J NeuroSci. 30: 11951
April 2013
Total internal reflection (TIRF) microscope
April 2013
Total internal reflection (TIRF) microscope
Advantages:
+ High signal to noise ratio
+ Very fast acquisition possible
+ Single molecule detection
+ Very good for studying vesicle-membrane fusion events and
cell adhesion
Disadvantages:
- Only fluorescence directly at cover slip
April 2013
Systems
Advantages
Disadvantages
Deconvolution
•High light efficiency
•Very fast frame rates possible
(CCD/sCMOS)
•Low depth discrimination
•Filterchanges usually slow
•Results only after deconvolution
CLSM
•High resolution
•Optical zoom
•Optical sectioning
•Low signal/noise
•Relative slow acquisition (PMT)
Spinning disk
•Optical sectioning less than
CLSM
•Higher frame rate than CLSM
(CCD/sCMOS)
•Multichannel usually sequential
•No optical zoom
•Less depth discrimination than
CLSM
MP LSM
•Optical sectioning without
pinhole
•High resolution
•Deep penetration (NIR)
•Only excitation at focal point
•Expensive
•One channel at a time
•Relative slow acquisition (PMT)
TIRF
•High signal/noise
•Very fast frame rates possible
(CCD/sCMOS)
•Single molecule detection
•Only fluorescence directly at cover
slip
Structured illumination
OptiGrid system
QiOptiq
April 2013
Structured illumination
Grid is only in focus in the focal plane of the sample
Three raw images are combined to one in which the grid lines and the out of focus signal
have disappeared due to some claver calculations. Contrast and image sharpness are
markedly improved
Carl Zeiss MicroImaging, Thornwood,
April 2013
Structured illumination
Advantages
+ Cheap
+ Can be added to any fluorescence microscope
+ Results are directly visible
Disadvantages
- Slow
No live cell imaging!
- Need bright sample, increased bleaching
April 2013
Light sheet microscopy
Huisken J , Stainier D Y R Development 2009;136:1963-1975
April 2013
Light sheet microscopy
Principle discribed in 1903, first system in 1993 by Voie et al.
(J. Microsc. 170: 229–236. ) orthogonal-plane fluorescence
optical sectioning (ORFOS)
 Selective plane illumination microscopy (SPIM)
 Digital scanned laser light sheet fluorescence microscopy
(DSLM)
 Thin laser light sheet microscope Light sheet microscopy
(TLSM)
April 2013
Light sheet microscopy
Reconstruction of Zebrafish Early Embryonic Development
Keller et al (2010) Nature methods
April 2013
Super resolution microscopy
Imaging with a resolution below the diffraction limit of light
 Structured illumination microscopy (SIM)
 Photo-activation localization microscopy (PALM)
 Stochastic optical reconstruction microscopy (STORM) and
3D STORM
 Stimulated Emission Depletion microscopy (STED)
 Ground State Depletion microscopy (GSD)
 4pi (taken of the market)
April 2013
Structured illumination microscopy (SIM)
April 2013
Structured illumination microscopy (SIM)
CLSM
SIM
XY-resolution 100 nm
Z- resolution 200-300 nm
• Still diffraction limited
• Normal dyes
• Two fold resolution improvement
April 2013
Structured illumination microscopy (SIM)
DeltaVision | OMX
3D-SIM™ Super-Resolution Imaging
April 2013
Stimulated Emission Depletion microscopy
(STED)
Fluorescence is completely
suppressed by stimulated
emission process.
This ring-shaped pulse effectively provides a tiny aperture
April 2013
Stimulated Emission Depletion microscopy
(STED)
Sieber et al. Science 317:
1072-1076
April 2013
Stimulated Emission Depletion microscopy
(STED)
Bückers et al., Opt. Express 19 (2011): 3130 - 3143
April 2013
Ground state depletion microscopy
photobleaching
April 2013
Ground state depletion microscopy
photobleaching
April 2013
Ground state depletion microscopy
Dark state of a fluorophore; higher energy state that does not omit light
500 frames; 22
frames/s
Q-dots
CellR system
Straatman
Olsen and McKenzie (2010) A dark excited state of fluorescent protein chromophores,
considered as Brooker dyes. Chemical Physics Letters 492: 150-156
April 2013
Ground state depletion microscopy
Ptk2-cells.
Anti-NUP153/Alexa FLUOR 532 and
anti-β-tubulin/Alexa FLUOR 488
Wernher Fouquet, Leica Microsystems, Anna
Szymborsak and Jan Ellenberg, EMBL,
Heidelberg, Germany.
According to Leica website: Acquisition time 2-10 minutes
Maximum resolution down to 20 nm
Standard fluorochromes can be used – no need to change your
protocols
April 2013
PALM/STORM
PALM: use photo-activatable dyes (e.g., paGFP)
STORM: need photo-switchable fluorochromes
Imaging laser (657 nm)
Activator
Cy3
Cy5
Reporter
6000
photons
Activation
Cy3
Cy5
Cy3
Cy5
Activation laser (532 nm)
April 2013
PALM/STORM/GSD
Bright field microscope
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
April 2013
PALM/STORM/GSD
Brightfield
STORM
April 2013
█ Cy3 / Alexa 647: Clathrin
█ Cy2 / Alexa 647: Microtubule
5 μm
Bates et al, Science 317, 1749 – 1753 (2007)
1 μm
And there is now also 3D STORM
200 nm
Resolvable volumes obtained with current commercial
super-resolution microscopes
© 2010 Schermelleh et al.
/GSD
Schermelleh L et al. J Cell Biol doi:10.1083/jcb.201002018
April 2013
Whole animal imaging systems
Multispectral imaging
March 2012
Whole animal imaging systems
A; Original RGB image
C; FITC
D; TRITC
E; Cy3.5
F; Food
G; Skin autofluorescence
H; Merge
Levenson and Mansfield, Cytometry A 2006
March 2012
http://zeiss-campus.magnet.fsu.edu/tutorials/basics/axioobserver/index.html
Light sheet microscopy
Reconstruction of Zebrafish Early Embryonic Development
H2B-eGFP mRNA injected in one-cell stage
Total about 400,000 images per embryo
Keller et al (2008) Science 322
April 2013