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Core Biotechnology Services (CBS) College of Medicine, Biological Sciences and Psychology
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
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
Renaissance in biological imaging
April 2013
Whole animal imaging systems
(Maestro, CRi) Super resolution microscopy;
STORM images of mammalian
mitochondria. Zhuang Research Group, Department of Chemistry and
Chemical Biology, Harvard University, Cambridge, MA
R. Weissleder, U. Mahmood and J. Tam,
Massachusetts General Hospital
Renaissance in biological microscopy
April 2013
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
Simple microscope
Van Leeuwenhoek’s microscope Around 1668
Hans Loncke
July 2007 Micscape Magazine
Two Trinacria species of about 0,1 mm size
April 2013
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)
Compound microscope
April 2013
Light source (laser) Camera
CO2 and temperature control Incubator
Joystick to move stage
Anti-vibration table
Compound microscope
April 2013
Aberrations
Around 1668
3-9x magnification
Up to 275x mag
1595
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
diffraction limited microscope
η = refractive index of medium η.sin = NA (numerical aperture)
April 2013
1872: Ernst Abbe formulates his ‘wave theory of microscopic imaging’ :
d = λ 2η sin η = refractive index of medium
η.sin = NA (numerical aperture)
Refractive index (η ): the light-bending ability of a medium.
d = 0.5 * λ NA
April 2013
Resolution
April 2013
Resolution
Diffraction limited microscope
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.
Resolution
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
400 nm 488 nm 633 nm Excitation:
0
50
100
150
200
250
300
15
36
57
78
99
12
0
14
1
16
2
18
3
20
4
22
5
24
6
26
7
28
8
30
9
33
0
633
488
400
d = 0.5λ /NA R = 0.61λ /NA
• Radial resolution:
Resolution
April 2013
Resolution
April 2013
Resolution
d = 2λη/(NA)2
Axial resolution:
λ XY (nm) Z (nm)
488 231 754
561 244 867
XY YZ
XZ
(NA = 1.4; η = 1.515)
XY (2D): pixel
XYZ (2D): voxel
April 2013
Objectives
d = 0.5 λ /NA
But magnification has influence on optimal pixel size of CCD camera. Size of field of view for a fixed CCD chip.
R = 0.61 λ /NA
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
Adapted from: http://biology.fullerton.edu/facilities/em/BrightSetup.html
•Centre the condenser •Open field diaphragm till whole view is filled
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
Hematoxylin stain (histology)
Solution 3: stain your sample with colour dyes.
Blue = DNA; Green = talin; Red = actin;
Solution 4: Fluorescence microscopy
No colour and too little contrast between structures with similar transparency.
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 :
luciferin + ATP + O2 Oxyluciferin + AMP + PPi + CO2 + light
(PPi = pyrophosphate)
Firefly Luciferase (reporter gene)
Emission peak ~ 560 nm
Coelenterazine + O2 Coelenteramide + CO2 + light
Emission peak ~ 480 nm
Renilla luciferase
April 2013
Bioluminescence
Kwon et al. (2010) BioTechniques 48: 460-462
The green-emitting luciferase was derived from a Japanese luminous beetle (λmax 560 nm); the red-emitting luciferase was derived from railroad worm (λmax = 630 nm)
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
Absorption of light with a short λ resulting in emission of light with a longer λ (The so-called Stokes shift) Jablonski diagram
April 2013
Fluorochrome: molecule that is fluorescent. Fluorophore: a component of a molecule which causes this molecule to be fluorescent.
Fluorescence microscopes
April 2013
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
Filters
April 2013
Old filter
Exciter ET470/40x •
• Dichroic T495LP
• Emitter ET525/50m
Filters
April 2013
New filter
Exciter s D350/50x; •
• Dichroic 62002BS
• Emitter 61002m
• S492/18x; • S572/23x
Filters
April 2013
Filters
Filter wheel
April 2013
Filters
Borlinghaus and Kuschel; Nature Methods - 3, (2006)
Prism based
diffraction grating April 2013
Arc bulb
Mercury Arc lamps (e.g. X-cite)
Xenon Arc lamps
Metal Halide Arc lamps
LED (light-emitting diode )
Monochromator
Laser
Light sources
April 2013
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
Light sources
Arc bulb
April 2013
Xenon is relatively weak but has a more continuous and uniform spectrum what makes it more preferable for quantitative imaging
Light sources
Arc bulb
April 2013
• 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
Light sources
LED
April 2013
Light sources
Monochromator
April 2013
• Single wavelength
• High power
• White laser (supercontinuum laser)
• A laser can be focused or non-focussed
Light sources
Laser
April 2013
CCD (charge-coupled device)
EM-CCD (electron-multiplying CCD)
CMOS (complementary-metal-oxide-semiconductor
detector); much higher readout speed.
PMT (photomultiplier tube)
Detectors
April 2013
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
Detectors
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
• 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
Detectors
Alternative: SNR = (Signal – Background)/Standard Deviation of Background
April 2013
Detectors
Read noise (fixed)
CCD read out
CMOS reads out pixel by pixel Fast Noise/pixel
April 2013
EM-CCD camera has an Amplification stap before output node More sensitive Extra noise (excess noise factor)
Output node
Coates et al (2009) Scientific CMOS Technology A High-Performance Imaging Breakthrough White Paper
Detectors Cooled to -70 to -90 °C Reduced dark current
April 2013
Noise limited image i.e. the signals below the noise cannot be seen
EMCCD: minimizing the read noise and dark current
Detectors
Photometrics
April 2013
EM-CCDs Hamamatsu
Detectors
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
Camera pixel size = 6.45x6.45 µm. So without much loss of resolution you could image with a 2x2 binning using the 100x objective
R = 0.61λ /NA with λ = 550nm
April 2013
Binning
Binning increases image intensity while decreasing resolution & transfer time
April 2013
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
Binning
Image Quality
The quality of a microscope image is assessed by the following: Noise – S/N Focus - Is the image blurry or well-defined?
Brightness - How light or dark is the image? Contrast - What is the difference in colour and light between adjacent areas of the specimen?
Resolution – What is the minimal distance between two points in the image that still can be seen as two separate points?
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, same exposure time
Increasing gain reduces the number of photons / gray scale value
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
Live cell imaging Multiple colours Time series Multiple position Multiple z-sections
Epi-fluorescence microscope
April 2013
Widefield PSF SVI
Deconvolution microscope
Using maths to remove haze and blur
April 2013
192 optical sections of a fruit fly embryo leg acquired in 0.4-micrometer z-axis steps with a widefield fluorescence microscope
Deconvolution microscope
April 2013
sample
Z-series
3D-reconstruction
First confocals in the 50s First commercial system in 1987
Confocal laser scanning microscope
April 2013
3D pixel = voxel
Blue = DNA Green = Centrosome Red = Microtubules
3D-reconstruction Optical sections
Confocal laser scanning microscope
April 2013
April 2013
Resolution CLSM
256x256 128x128 512x512 1024x1024
April 2013
Deconvolution
Confocal laser scanning microscope
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
PSF
XY
Z
widefield
Spinning disk
CLSM
Stefan Terjung, EMBL
Spinning disk microscope
April 2013
Multi-photon laser scanning microscope
April 2013
April 2013
Multi-photon laser scanning microscope
Even scattered fluorescence photons are usefull in 2-photon 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
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
CLSM (50 µm)
Mouse cortical pyramidal neurons expressing EYFP. Excitation using 920 nm. (Zeiss LSM 7 MP brochure)
Multi-photon laser scanning microscope
April 2013
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
Total internal reflection (TIRF) microscope
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
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,
Grid is only in focus in the focal plane of the sample
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
Structured illumination
Light sheet microscopy
Huisken J , Stainier D Y R Development 2009;136:1963-1975
April 2013
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
Keller et al (2010) Nature methods
Reconstruction of Zebrafish Early Embryonic Development
April 2013
Light sheet 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)
Super resolution microscopy
April 2013
Structured illumination microscopy (SIM)
April 2013
XY-resolution 100 nm Z- resolution 200-300 nm • Still diffraction limited • Normal dyes • Two fold resolution improvement
SIM
Structured illumination microscopy (SIM)
CLSM
April 2013
DeltaVision | OMX 3D-SIM™ Super-Resolution Imaging
April 2013
Structured illumination microscopy (SIM)
Fluorescence is completely suppressed by stimulated emission process.
This ring-shaped pulse effectively provides a tiny aperture
Stimulated Emission Depletion microscopy (STED)
April 2013
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
Stimulated Emission Depletion microscopy (STED)
photobleaching
Ground state depletion microscopy
April 2013
photobleaching
April 2013
Ground state depletion microscopy
Dark state of a fluorophore; higher energy state that does not omit light
Olsen and McKenzie (2010) A dark excited state of fluorescent protein chromophores,
considered as Brooker dyes. Chemical Physics Letters 492: 150-156
500 frames; 22
frames/s
Q-dots
CellR system
Straatman
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: Maximum resolution down to 20 nm
Standard fluorochromes can be used – no need to change your protocols
Acquisition time 2-10 minutes
April 2013
Ground state depletion microscopy
PALM: use photo-activatable dyes (e.g., paGFP) STORM: need photo-switchable fluorochromes
Activation
Imaging laser (657 nm)
Activation laser (532 nm)
Cy3 Cy5
Cy3 Cy5 Cy3 Cy5
Activator Reporter
6000 photons
PALM/STORM
April 2013
Bright field microscope
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
April 2013
STORM Brightfield
PALM/STORM/GSD
April 2013
5 μm
█ Cy3 / Alexa 647: Clathrin
█ Cy2 / Alexa 647: Microtubule
Bates et al, Science 317, 1749 – 1753 (2007)
1 μm
200 nm
And there is now also 3D STORM
Resolvable volumes obtained with current commercial super-resolution microscopes
Schermelleh L et al. J Cell Biol doi:10.1083/jcb.201002018
© 2010 Schermelleh et al.
/GSD
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
Keller et al (2008) Science 322
Reconstruction of Zebrafish Early Embryonic Development
H2B-eGFP mRNA injected in one-cell stage Total about 400,000 images per embryo
April 2013
Light sheet microscopy
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