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Transcript of 3D light microscopy techniques - ZMB Deconvolution techniques: ¢â‚¬¢2D...

  • 3D light microscopy techniques

  • The image of a point is a 3D feature

    In-focus image

    Out-of-focus image

  • 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-focus part

    Out-of-focus part

  • Result is a blurred image with 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 the arc lamp

    PMT replaces the CCD 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 sectioning point-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

    10ns 100fs

    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 scattering of excitation rays (long wavelength)

    Capture of scattered, emitted rays

  • 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 imaging Long duration live cell microscopy Lower resolution compared to confocal

    – Long wavelength excitation Thermal damage from chromophores that absorb in

    the IR spectrum Dependent on fluorescence Expensive (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 object distribution

    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 Cell Exp: 0.5 s Lens: 100x/1.4

  • Microtubules in Toxoplasma gondii in the WF Microscope

    Raw Data

    Decon’d Ke Hu David Roos John 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

    3D light microscopy techniques The image of a point is a 3D feature The image of a point is not a point Resolution is now an arbitrary measure of how close two point images can come such that they are perceived as separate Image formation in a light microscope The role of the OTF (or MTF) 3D Information transfer In a 3D object we have cross-talk between in- and out-of-focus parts Result is a blurred image�with substantial background intensity Reduce out-of-focus information by inserting a pinhole Result: much sharper pictures In practice, confocal microscopes are point scanners Thick sample imaging Image formation in the confocal microscope Confocal vs widefield microscope Multi-photon microscopy Fluorescence fundamentals 2 photon microscopy 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 Demonstration of 2-photon performance on a pollen grain Major advantages (and usefulness) Summary: multiphoton microscopy Selective plane illumination microscopy Numerous SPIM versions exist Long term SPIM imaging – Tomancak lab SPIM limitations The triangle of compromises Deconvolution Deconvolution microscopy:�the alternative for rapid 3D imaging The mathematical challenge Deconvolution One possible approach:�iterative deconvolution Widefield, deblurring, full deconvolution Slide Number 35 Slide Number 36 Conclusion: Confocal vs. deconvolution microscopy