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