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Biophotonics lecture 23. November 2011

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Last week: -Fluorescence microscopy in general, labeling, etc -How to do optical sectioning and fill the missing cone, the confocal microscope

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Today: -Structured illumination: an alternative approach to optical sectioning -Super resolution techniques: beyond the Abbe limit -High-resolution structured illumination microscopy

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Optical sectioning alternatives: structured illumination microscopy (SIM) -Sample is illuminated with a structured illumination pattern, i.e. a line grid. -This can be produced by placing a grid in a conjugate image plane in the illumination pathway. -Three images are taken for three different (lateral) positions of the illumination grid. -These images can be computed into a final, optically sectioned image.

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For single-shot sectioning

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In-focus sample slice Out-of-focus sample slice Full-field illumination Structured illumination Acquired wide-field image Acquired structured illumination image x y x z x y x y

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Acquired structured illumination images I1(r)I1(r)I2(r)I2(r)I3(r)I3(r) I final (r) = max { I 1 (r), I 2 (r), I 3 (r) } - min { I 1 (r), I 2 (r), I 3 (r) } Result: sectioned imageWide-field image

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Advantages: -Cheaper: no need for laser, scanner, PMTs, AOTFs, etc. -Potentially faster: for a large field of view 3 wide-field images can be acquired faster than a point-wise scan. -Less photo-bleaching: less light is lost as compared to using a pinhole

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Wide-field Sectioning SIM Frequency support of sectioning SIM -Filled missing cone -Higher axial support -Higher lateral support (1 direction), more on this later But this is NOT an OTF !!

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Wide-field Confocal Sectioning SIM The image formation in section SIM cannot be written as a convolution of the sample with an intensity PSF.

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Advantages: -Cheaper: no need for laser, scanner, PMTs, AOTFs, etc. -Potentially faster: for a large field of view 3 wide-field images can be acquired faster than a point-wise scan. -Less photo-bleaching: less light is lost as compared to using a pinhole Disadvantage: -Not a linear imaging modality. Not as useful for quantitative analysis.

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Imaging beyond the Abbe-limit: the true strength of SIM

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Sample for simulation Fourier transform of Sample Sample will be repainted with a blurry brush rather than a point-like brush. Real space Fourier space Limited resolution in conventional, wide-field imaging

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Moir effect high frequency detail high frequency grid low frequency moir patterns

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Moir effect Structured Illumination Microscopy Illumination with periodic light pattern down-modulates high- frequency sample information and makes it accessible for detection. SampleIllumination

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Laser CCDxz Tube lens Filter Dichromatic reflector Tube lens Objective Sample Diffraction grating, SLM, etc

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SampleIllumination Structured Illumination Micropscopy Sample with structured illumination Multiplication of sample and illumination

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Structured Illumination Micropscopy Multiplication of sample and illumination Real space Fourier space Convolution of sample and illumination Convolution of sample and illumination

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SampleIllumination Structured Illumination Micropscopy

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Sample

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Sample Sample & llumination

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Sample Imaging leads to loss of high frequencies (OTF) Structured Illumination Micropscopy

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Separating the components Sample Structured Illumination Micropscopy

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Separating the components Shifting the components Sample Structured Illumination Micropscopy

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Separating the components Shifting the components Recombining the components Sample Structured Illumination Micropscopy

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Separating the components Shifting the components Recombining the components using the correct weights. Sample Reconstructed sample Structured Illumination Micropscopy

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samplewide-field SIM (x only)

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Missing cone no optical sectioning Full-field illumination 1 focus in back focal plane

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Missing cone no optical sectioning 2-beam structured illumination 2 foci in back focal plane

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Missing cone filled optical sectioning 2-beam structured illumination 3 foci in back focal plane better z-resolution

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1 m Fourier space (percentile stretch) Liisa Hirvonen, Kai Wicker, Ondrej Mandula, Rainer Heintzmann

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WF: 252 nm SIM: 105 nm 99 beads averaged wide-field SIM

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2 m excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris Samples Prof. Bastmeyer, Universitt Karlsruhe (TH) Axon Actin (Growth Cone)

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excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris 2 m Samples Prof. Bastmeyer, Universitt Karlsruhe (TH) Axon Actin (Growth Cone)

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Doublets in Myofibrils Isolated myofibrils from rat skeletal muscle Titin T12 Oregon green L. Hirvonen, E. Ehler, K. Wicker, O. Mandula, R. Heintzmann, unpublished results 1 m 124 nm

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1 m Molecules in space and time living COS1 cell L. Hirvonen, K. Wicker., O. Mandula and R. Heintzmann, Structured illumination microscopy of a living cell, Europ. Biophys. J. 38, 807-812, 2009

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Marie Walde, James Monypenny (cooperation G. Jones), Kings College London f-Actin Vinculin Are podosomes arranged as "sticks and joints"? 5 mPodosomes

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0 magnitude spatial frequency Support region of OTF 0 magnitude spatial frequency -K 0 K0K0 -2K 0 -K 0 K0K0 Linear Excitation (low intensity) Non-Linear Excitation (high intensity) -3K 0 Support region of OTF

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Conventional microscopy Saturated structured illumination 1 m Linear structured illumination 1 m Mats Gustafsson, UCSF 50 nm microscpheres nonlinearity: fluorescence saturation, 53J/m 2 3 extra harmonics M.G.L. Gustafsson (2005), PNAS, 37, 13081-13086 Nonlinear Structured Illumination Micropscopy

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But: Artefacts possible! 0.5 m

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But: Artefacts possible! Sophisticated algorithms are needed!