Advanced Optical Microscopy lecture. 03. December 2012 Kai Wicker
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1 Advanced Optical Microscopy lecture 03. December 2012 Kai Wicker
2 Today: Optical transfer functions (OTF) and point spread functions (PSF) in incoherent imaging. 1. Quick revision: the incoherent wide-field OTF (and the missing cone) 2. Filling the missing cone: the confocal microscope 3. Imaging through two objectives: the 4Pi microscope 4. Superresolution imaging: Structured illumination microscopy (SIM)
3 1. Quick revision: the incoherent wide-field OTF (and the missing cone)
4 McCutchen generalised aperture 2pn/l Ewald sphere
5 Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy Missing cone Axial support Lateral support
6 The missing cone and optical sectioning: Missing cone Axial support Lateral support
7 2. Filling the missing cone: the confocal microscope
8 The confocal microscope
9
10
11 The confocal PSF: Point spread function, PSF: - Wide-field imaging: The image generated by a point-source. Or: - Scanning: The amount of signal detected from a point-source in dependence on the source s position.
12 The confocal PSF: Sample scan scan position: s Let s first look at the detection only (i.e. constant illumination everywhere): - The sample is a point source, shifted to the scan position s. - The point source emits light, which forms an image ( PSF around the position s) - in the image plane: h emission (r s) - Light not falling onto the (centered) pinhole p r is blocked. Right behind the pinhole the light distribution is: h emission r s p r = h emission s r p r with h em. r :=h em. r - The resulting light distribution is integrated on the PMT detector, yielding the final confocal PSF: h detection s = h emission s r p r dr = h emission p (s) - The detection PSF is a convolution of the (mirrored) emission PSF with the pinhole.
13 The confocal PSF: Sample scan scan position: s Let s combine this with point scanning illumination: - The illumination is centered, i.e. fixed at position 0. The shape of the illumination is given by the illumination PSF: h illumination (r) - The point source at position s is illuminated with light of brightness: h illumination (s) - The detection signal h detection s has to be scaled with this brightness: h illumination (s)h detection s - This combined signal is the confocal PSF: h confocal s = h illumination (s)h detection s h confocal s = h illumination (s) h emission p (s)
14 Confocal fluorescence microscopy Reduction of out of focus light Resolution in confocal microscopy Comparison of axial (x-z) point spread functions for widefield (left) and confocal (right) microscopy
15 The confocal OTF: PSF(r) = PSF Excitation (r) PSF Detection (r) OTF(k) = OTF Excitation (k) OTF Detection (k) Missing cone has been filled!! Lateral support has been increased. Missing cone k z Axial support has been increased. k x,y a k x,y Increasing the aperture angle (a) enhances resolution!! k z
16 Top view Missing cone
17 We have circumvented the Abbe-limit, BUT: WF Limit in-plane, in-focus OTF 1.4 NA Objective 1 AU WF 0.3 AU Almost no transfer New Confocal Limit
18 Wide-field vs. confocal Widefield Mouse Brain Hippocampus Smooth Muscle Sunflower Pollen Grain Confocal Comparison of widefield (upper row) and laser scanning confocal fluorescence microscopy images (lower row). (a) and (b) Mouse brain hippocampus thick section treated with primary antibodies to glial fibrillary acidic protein (GFAP; red), neurofilaments H (green), and counterstained with Hoechst (blue) to highlight nuclei. (c) and (d) Thick section of rat smooth muscle stained with phalloidin conjugated to Alexa Fluor 568 (targeting actin; red), wheat germ agglutinin conjugated to Oregon Green 488 (glycoproteins; green), and counterstained with DRAQ5 (nuclei; blue). (e) and (f) Sunflower pollen grain tetrad autofluorescence.
19 3. Imaging through two objectives: the 4Pi microscope Filling (part of) the missing cone by enlarging the NA.
20 Aperture increase: 4 Pi Microscope (Type C) Sample between Coverslips Stefan W. Hell Max Planck Institute of Biophysical Chemistry Göttingen, Germany z Detector Pinhole Fluorescence Intensity Dichromatic Beamsplitter Illumination Emission 2 Photon Effect High Sidelobes z
21 ATF OTF widefield 4Pi?
22 4Pi PSFs widefield, l=500nm 4Pi, l=500nm
23 Leica 4Pi Image:
24 4Pi images Deviding Escherichia Coli Axial direction From: Bahlmann, K., S. Jakob, and S. W. Hell (2001). Ultramicr. 87:
25 4. Superresolution imaging: Structured ilumination microscopy (SIM)
26 Limited resolution in conventional, wide-field imaging Real space Fourier space Sample for simulation Fourier transform of Sample Sample will be repainted with a blurry brush rather than a point-like brush.
27 Moiré effect high frequency detail high frequency grid low frequency moiré patterns
28 Moiré effect Illumination Sample Structured Illumination Microscopy Illumination with periodic light pattern down-modulated highfrequency sample information and makes it accessible for detection.
29 CCD x Laser Tube lens Tube lens Filter Dichromatic reflector z Diffraction grating, SLM, etc Objective Sample
30 Structured Illumination Micropscopy Sample Sample with structured illumination Illumination Multiplication of sample and illumination
31 Structured Illumination Micropscopy Real space Fourier space Convolution of sample and illumination Multiplication of sample and illumination
32 Structured Illumination Micropscopy Sample Illumination
33 Structured Illumination Micropscopy Sample
34 Structured Illumination Micropscopy Sample Sample & llumination
35 Sample Sample & llumination Imaging leads to loss of high frequencies (OTF)
36 Sample Separating the components
37 Sample Separating the components Shifting the components
38 Sample Separating the components Shifting the components Recombining the components
39 Sample Reconstructed sample Separating the components Shifting the components Recombining the components using the correct weights.
40 sample wide-field SIM (x only)
41 Missing cone no optical sectioning 1 focus in back focal plane Full-field illumination
42 Missing cone no optical sectioning 2 foci in back focal plane 2-beam structured illumination
43 Missing cone filled optical sectioning 3 foci in back focal plane better z-resolution 2-beam structured illumination
44 Fourier space (percentile stretch) 1 mm Liisa Hirvonen, Kai Wicker, Ondrej Mandula, Rainer Heintzmann
45 99 beads averaged WF: 252 nm SIM: 105 nm wide-field SIM
46 2 µm Axon Actin (Growth Cone) excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris Samples Prof. Bastmeyer, Universität Karlsruhe (TH)
47 2 µm Axon Actin (Growth Cone) excite 488nm, detect > 510 nm 24 lp/mm = 88% of frequency limit Plan-Apochromat 100x/1.4 oil iris Samples Prof. Bastmeyer, Universität Karlsruhe (TH)
48 Doublets in Myofibrils 1 µm 124 nm Isolated myofibrils from rat skeletal muscle Titin T12 Oregon green L. Hirvonen, E. Ehler, K. Wicker, O. Mandula, R. Heintzmann, unpublished results
49 3d live cell SIM cytosol (green), actin (red) Images by Reto Fiolka, Janelia Farm Research Campus, HHMI, Ashburn, VA, USA
50
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