3d light microscopy techniques - zmb uzh€¦ · deconvolution techniques: •2d...
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3D light microscopy techniques
The image of a point is a 3D feature
In-focusimage
Out-of-focusimage
The image of a point is not a point
Point Spread Function (PSF)
1D imaging
2D imaging
3D imaging
Resolution is now an arbitrary measure of how close two point images can come such that they are
perceived as separate
Lord Rayleigh’s criterion:
λ = 488 nm (NA = 1.4) → δ = 212 nm; δz= 780 nm
(NA = 0.4) → δ = 744 nm; δz= 9.56 micron
Image formation in a light microscope
∫∞
∞−
+−Ψ=Φ )()()()( xnydyxPSFyx n – noise
The role of the OTF (or MTF)
3D Information transfer
• In analogy to the twodimensional image formation, we can determine a 3D Point spread function (PSF) and a 3D Optical Transfer function (OTF).
kzzPSF OTF
z=0 z=2µm
In a 3D object we have cross-talk between in- and out-of-focus parts
In-focuspart
Out-of-focuspart
Result is a blurred imagewith substantial background intensity
Reduce out-of-focus information by inserting a pinhole
emission pinholeIllumination /exitation pinhole
confocal planes
Result: much sharper pictures
non-confocal = wide-field
confocal
In practice, confocal microscopes are point scanners
Laser replaces thearc lamp
PMT replaces theCCD camera
Thick sample imaging
Image formation in the confocal microscope
Again the image is formed by a convolution, but the confocal PSF is smaller and has no „butterfly wings“.
The optical transfer function has an ellipsoidal shape and has no discontinuity in the middle- optical sectioning
z
Widefield PSF confocal
confocal OTF
kz
Confocal vs widefield microscope
sharp optical sectioningpoint-scanning method (slow)majority of returned photons not detected
– wait for a long time to get robust signal• even slower• Photodetector noise gets critical (weak SNR)• Photodamage on sample
nice additional features: use programmability of laser scans – for bleaching experiments– for selective point measurements in small volumes
(spectroscopy, fluorescence correlation spectroscopy)
Multi-photon microscopy
Fluorescence fundamentals
2 photon microscopy
10ns100fs
Because of the extremly high photon density at the focal point, it is possible that two photons interact simultaneously with a fluorophore.
No bleaching in the out-of-focus planes, but increased photo-bleaching in the focal plane (~10faster)!
Pulsed lasers (typically Ti:Saph ) and tight focusing increase the photon flux.
Linescan using confocal and 2 photon microscopylens
The multi-photon microscope(in comparison to conventional and confocal microscopy)
The major advantage is the ability to reduce the influence of light scattering in the sample
Scattering of excitation rays
Scattering of emitted rays
Less scatteringof excitation rays(long wavelength)
Capture of scattered, emittedrays
Demonstration of 2-photon performance on a pollen grain
20 µm
Major advantages (and usefulness)
• Imaging of scattering samples– Deep sections and whole tissue imaging
• Maximal use of light– Shorter exposure times and levels
• Low photobleaching outside the focal volume– Long observation possible– Low photo-toxicity
Summary: multiphoton microscopy
Thick section imagingLong duration live cell microscopyLower resolution compared to confocal
– Long wavelength excitationThermal damage from chromophores that absorb in
the IR spectrumDependent on fluorescenceExpensive (requires a pulsed laser setup)
Selective plane illumination microscopy
Re-discovering a 100 years old idea…
• Fast (camera-based)• Inherent sectioning capability (like a confocal), without
«throwing» light away• Rotation of the sample: uniform imaging (resolution) – like
tomography• Minimized bleaching/photo-toxicity (only the interesting
plane is illuminated)
Numerous SPIM versions exist
Long term SPIM imaging – Tomancak lab
SPIM limitations
• Sample size (thickness)• Sample mounting• Aberrations• Data amount
The triangle of compromises
Signal/Noise ratio
(image quality)
Imaging speedImage resolution
Deconvolution
=*
Image is formed by convolution of the 3D Object with the PSF. Can this opertation be inverted?
Deconvolution microscopy:the alternative for rapid 3D imaging
measured image
measured PSF
modeled PSF
unknown PSF
unknown objectdistribution
unknown noise
The mathematical challenge
A simple idea:
Two practical difficulties:1.) H(ν) is not always positive (bandpass and aberrations)2.) Noise in I(ν) gets amplified by division by small H(ν)
−= ∫
∞
∞−
ydyxhygxiyg )()(),(bestmatch)(ˆ
Deconvolution
• The inverse operation of the convolution, the deconvolution, is the division of the image spectrum with the OTF. Division by nearly zero and zero is not such a good idea.
• No information has been physically transfered outside of the support of the OTF (nonzero region), so no information can be reconstructed. Still people try it and corresponding software has become available.
Deconvolution techniques:
•2D Methods:Deblurring: simply subtracts estimate of out of focus light
•3D-Methods: Image Restoration, tries to reassign out of focus light to its source
One possible approach:iterative deconvolution
Close ?
YES
NO
Widefield, deblurring, full deconvolution
Widefield, deblurring, restoration
Restored
Unprocessed
Nearest Neighbor
• Both deblurring and restoration improve contrast• SNR significantly lower for deblurred image• Deblurring results in loss of pixel intensity• Restoration results in gain of pixel intensity
XLK2 CellExp: 0.5 sLens: 100x/1.4
Microtubules in Toxoplasma gondii in the WF Microscope
Raw Data
Decon’dKe HuDavid RoosJohn Murray
© Jason Swedlow 2001
Conclusion: Confocal vs. deconvolution microscopy
• Confocal is the optimal 2D microscope!• Deconvolution microscopy is the faster
technique in 3D (in acquisition – not in data analysis)
• Where affordable: combine confocal and deconvolution microscopy for optimal 3D imaging