Lec5_MP_SR_Microscopy_WZ

Lec5_MP_SR_Microscopy_WZ - Part 1: Laser-scanning...

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1 Part 1: Laser-scanning microscopy - Multiphoton microscopy Part 2: Super-resolution microscopy AEP 470 – BIONB 470 – BMEP 570–VETPR 470 Biophysical Methods Sept. 18, 2008 Warren R. Zipfel [email protected] Department of Biomedical Engineering Developmental Resource for Biophysical Imaging and Opto-electronics Applied and Engineering Physics Cornell University, Ithaca, NY 14853 Total magnification = Objective mag x eyepiece mag Focal length of an infinity corrected objective = f tube lens / Mag Tube lens focal length depends on the manufacturer Zeiss: f = 160 mm Olympus: f = 180 mm Nikon: f = 200 mm Entrance pupil diameter: 22 tubelens obs f EPD NAf NA Mag == Review of a conventional microscope: NA = n sin( α ) α # #2 11 2 (2 ) 1 NA f f =≈ + In terms of the “f-number” (=f/D) The point spread function (PSF) of a lens system is the image of an object that is much smaller than the illuminating wavelength. It is a mathematical function in 1, 2 or 3 dimensions that maps the intensity in image space: 1D 2D (along optical axis) 3D (xy plane, stepping in z) The illumination point spread function (IPSF) is what the focused illumination “looks like” near the focus.
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2 The common equations for the diameter of the central lobe of the PSF: Lateral FWHM = 0.51 λ / NA Axial FWHM = 1.77n λ / NA 2 Paraxial approach begins to break down above NA’s of ~0.7 and is most severe in the axial direction. Sheppard and Matthews (1987) developed a “pseudoparaxial” theory which is in better agreement with the full vectorial diffraction calculation of Richards and Wolf (1959). Axial FWHM = 0.44 λ / (n sin 2 ( θ /2)) (where θ = asin(NA/n)) Sheppard and Mathews (1987) J. Opt. Soc Am A, 4(8) 1354 Richards and Wolf . (1959) Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system. Proc. Roy. Soc. A 253: 358-379.) 2 NA λ ⎛⎞ ⎜⎟ ⎝⎠ 2 2 NA Lateral (xy) Axial (z) For a diffraction-limited focus the objective must be uniformly illuminated with a plane wave at the back aperture. Lateral direction Axial direction β = lens EPD/ 1/e beam diameter β = 4 β = 2 An image is a superposition of light from all points on the object in the image plane. ( ', ', ') ( ', ', ') ( , , ) all space image x y z psf x x y y z z object x y z dxdydz =− ∫∫∫ An image is a convolution:
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3 Optical Sectioning in Biological Microscopy Widefield Fluorescence Multiphoton Microscopy Confocal Microscopy Deconvolution Methods (Computational) Confocal Aperture nonlinear processes Widefield Fluorescence Fixation and Physical Sectioning Live specimens Conventional light microscopy doesn’t work well on thick (> few microns) specimens Fluorescently labeled sea urchin eggs 3D Images Z-series: acquired by stepping the focus through the specimen. 1. Projections: (a) Average projection - Average of pixel intensities in plane perpendicular to viewer (b) Maximum intensity projection - Maximum pixel value displayed in plane perpendicular to viewer 2. Isosurface rendering - surfaces are created connecting a selected pixel value.
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This note was uploaded on 03/29/2009 for the course A&EP 470 taught by Professor Lindau during the Fall '08 term at Cornell.

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Lec5_MP_SR_Microscopy_WZ - Part 1: Laser-scanning...

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