Introduction to Computational Fluorescence Microscopy EE367/CS448I: - - PowerPoint PPT Presentation

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Introduction to Computational Fluorescence Microscopy EE367/CS448I: - - PowerPoint PPT Presentation

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Introduction to Computational Fluorescence Microscopy EE367/CS448I: Computational Imaging and Display stanford.edu/class/ee367 Lecture 13 Gordon Wetzstein Stanford University Midterm Wednesday, Feb 26, 3-4:20 pm in Thornt 102 In

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Introduction to Computational Fluorescence Microscopy

Gordon Wetzstein Stanford University EE367/CS448I: Computational Imaging and Display stanford.edu/class/ee367 Lecture 13

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Midterm

  • Wednesday, Feb 26, 3-4:20 pm in Thornt 102
  • In

In-class ss – yo you need to be here!

  • pen book: use slides, internet, bring computer, whatever you like
  • can all be solved without programming, similar to theoretical questions
  • f the assignments
  • nly SCPD students can do remotely, we will be emailing you
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  • H. Rankin, transgenic xenopus laevis (african clawed toad) tadpole neurons (green); technique: confocal 10x
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  • M. Kandasamy, stained cells: actin (pink), DNA (yellow), mitochondria (green); technique: super resolution microscopy
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SLIDE 5
  • M. Boyle, larva of nephasoma pellucidum (peanut worm); technique: confocal 40X
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SLIDE 6
  • D. Burnette, osteosarcoma cell (bone cancer) showing actin (purple), mitochondria (yellow), DNA (blue); technique: structured illumination microscopy (SIM)
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SLIDE 7
  • T. Deerinck, HeLa cells with microtubules; technique: 2-photon microscopy 300X
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SLIDE 8

Nikon Small World Competition

  • annual photography competition, see www.microscopyu.com/smallworld/gallery/
  • showed only fluorescent samples (many others in the gallery)
  • this lecture: overview of fluorescence microscopy techniques
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SLIDE 9
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source: white house & nature

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Brain Initiative

  • frontier of science (past frontiers: fly to moon, decode human genome)
  • two key factors: fluorescence microscopy & computational illumination
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Deisseroth Lab, Stanford; CLARITY; Nature 2013

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SLIDE 13

Widefield Microscopy

source: microscopyu

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Microscope Objective

source: Zeiss

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The Diffraction Limit

Ernst Abbe, 1905

source: wikipedia

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The Diffraction Limit d = λ 2nsinα = λ 2NA α

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The Diffraction Limit d = λ 2nsinα = λ 2NA α

λ

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The Diffraction Limit d = λ 2nsinα = λ 2NA α

λ

Airy disk

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The Diffraction Limit d = λ 2nsinα = λ 2NA

Airy disk Rayleigh Criterion

source: wikipedia

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Lateral and Axial Resolution & Missing Cone

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Fluorescence Microscopy

  • excitation and emission
  • coherence / incoherence
  • fluorescent labels
  • calcium imaging
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Fluorescence Microscopy (epi setup)

source: wikipedia

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Fluorescence Microscopy (epi setup)

source: wikipedia source: Nikon MicroscopyU

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Sensors used in Microscopy

  • e.g., Andor iXon Ultra 897: cooled to -100° C or Hamamatsu Ocra

Flash4.0 V2

  • scientific CMOS & CCD (~20-50K)
  • reduce pretty much all noise, except for photon or shot noise
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Fluorescence Microscopy - Challenges

  • inherently 2D – need 3D for active brain imaging
  • higher-resolution in 2D and 3D
  • scattering
  • larger fields of view, bleaching
  • solution: engineer detection and illumination optics, algorithms, chemistry
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Fluorescence Microscopy - Challenges

  • inherently 2D – need 3D for active brain imaging
  • higher-resolution in 2D and 3D
  • scattering
  • larger fields of view, bleaching
  • solution: engineer detection and illumination optics, algorithms, chemistry
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SLIDE 27

Superresolution Fluorescence Microscopy

  • stimulated emission-depletion (STED) microscopy
  • localization microscopy
  • 2D: STORM/PALM etc.
  • 3D: double helix PSF
  • localization algorithms
  • structured illumination microscopy (SIM)
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2014 Nobel Price in Chemistry: super-resolved fluorescence microscopy

Eric Betzig (Howard Hughes Institute) Stefan Hell (Max Planck Institute)

  • W. E. Moerner

(Stanford)

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Stimulated Emission-Depletion (STED) Microscopy

source: wikipedia excitation spot de-excitation spot emitted spot

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Stimulated Emission-Depletion (STED) Microscopy

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Localization Microscopy: PALM / STORM

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Structured Illumination Microscopy (SIM)

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3D Fluorescence Microscopy

  • confocal microscopy
  • 2 photon microscopy
  • light sheet microscopy
  • 3D deconvolution microscopy / focal stacks
  • thers: spinning disk confocal, aperture correlation, …
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Confocal Microscopy

1957

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Confocal Microscopy

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Widefield vs Confocal – Thin Sample

source: http://microscopysolutions.ca/

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Widefield vs Confocal – Thick Sample

source: http://microscopysolutions.ca/

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2-Photon Microscopy

Denk et al. “Two-photon laser scanning fluorescence microscopy”, Science 1990; photo: microscopy.berkeley.edu

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2-Photon Microscopy

Denk et al. “Two-photon laser scanning fluorescence microscopy”, Science 1990; photo: microscopy.berkeley.edu

good scattering properties deep imaging

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3D Deconvolution Microscopy

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3D Deconvolution Microscopy

… whiteboard …

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Light Sheet Microscopy

Huisken et al. “Selective Plane Illumination Techniques in developmental biology”, Development 2009

  • invented by R.

Zsigmondy, Nobel price in 1925

  • Nature Method of the

Year 2014

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Ahrens et al. “Whole-brain functional imaging at cellular resolution using light-sheet microscopy”, Nature Methods 2013

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Light Field Microscopy

[Levoy et al. 2006]

  • can do refocus, but more interesting:

instantaneous 3D volume (for fluorescence)!

  • diffraction becomes an issue
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Light Field Microscopy

Levoy Group, Stanford

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Levoy Group, Stanford

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Light Field Microscopy

Levoy Group, Stanford

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3D Light Field Deconvolution

  • light field contains aliasing
  • use 3D deconvolution to

get higher resolution

[Broxton et al. 2013]

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3D Light Field Deconvolution

  • lateral resolution is depth dependent!

[Broxton et al. 2013]

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Functional 3D Brain Imaging

  • C. elegans

[Prevedel et al. 2014]

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Functional 3D Brain Imaging

Captured Light Field

  • ptics design by Marc Levoy

[Prevedel et al. 2014]

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maximum intensity projection of volume 350um x 350 um x 24 um at 50Hz ~70 neurons in head region

[Prevedel et al. 2014]

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[Prevedel et al. 2014]