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Molecular Cell Biology

Molecular Cell Biology. Light Microscopy in Cell Biology Cooper Modified from a 2010 lecture by Richard McIntosh, University of Colorado. Images from a light microscope can be strikingly informative about cells. How are these images made? What questions can they answer?

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Molecular Cell Biology

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  1. Molecular Cell Biology Light Microscopy in Cell Biology Cooper Modified from a 2010 lecture by Richard McIntosh, University of Colorado

  2. Images from a light microscope can be strikingly informative about cells How are these images made? What questions can they answer? What are their limitations? Can you make and use them?

  3. Scales of absolute size: powers of 10

  4. Light behaves as a Wave Wavelength sets limits on what one can see

  5. θ θ θ Lower limits on spatial resolution aredefined by the Rayleigh Criterion Resolution = 0.61 x wavelength of light NA (numerical aperture) NA = nsinθ n = refractive index of the medium θ = semi-angle of an objective lens The effect of NA on the image of a point. The need for separation to allow resolution

  6. Contrast in the Image is Necessary:Types of Optical Microscopy Generate Contrastin Different Ways • Bright field - a conventional light microscope • DIC (Differential Interference Contrast - Nomarski) • Phase contrast • Fluorescence • Polarization • Dark field

  7. Bright-field Optics: Light Passing Straight Through the Sample • Most living cells are optically clear, so stains are essential to get bright field contrast • Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable • Fixing and staining is an art

  8. Classic drawings and modern images made from Giemsa-stained blood smears Plasmodium falciparum Histidine-rich Protein-2

  9. Generating Contrast • Staining • Coefficients of absorption among different materials differ by >10,000, so contrast can be big • Without staining • Everything is bright • Most biological macromolecules do not absorb visible light • Contrast depends on small differences between big numbers • Need an optical trick

  10. Mammalian Cell: Bright-field and Phase-contrast Optics

  11. Principles of bright fieldand phase contrast optics

  12. Differential Interference Contrast (DIC) • Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen • Small amounts of contrast can be expanded electronically • Lots of light: Video camera with low brightness & high gain

  13. Brightfield vs DIC

  14. DIC has shallow depth-of-field:Image a single plane in a large object Worm embryo

  15. DIC: Good contrast. Detection vs Resolution.Microtubules: 25 nm diameter (1/10 res.lim.) but visible in DIC

  16. Fluorescent staining:High signal-to-noise ratio (white on black)

  17. Principle of Fluorescence • Absorption of high-energy (low wavelength) photon • Loss of electronic energy (vibration) • Emission of lower-energy (higher wavelength) photon

  18. Design of a Fluorescence Microscope

  19. Fluorescent tubulin injected into aDrosophila embryo, plus a DNA stain

  20. Green Fluorescent Protein - Considerations • Color - Not just green • Brightness • Time for folding • Time to bleaching

  21. Live-cell Imaging of Microtubule Ends: EB1-GFP chimera

  22. GFP-Cadherin in cultured epithelial cells

  23. Immunofluorescence • Primary Abs recognize the antigen (Ag) • Secondary Abs recognize the primary Ab • Secondary Abs are labeled

  24. Immunofluorescence Example • Ab to tubulin • Ab to kinetochore proteins • DNA stain (DAPI)

  25. Biological microscopy problem: Cells are 3D objects, and pictures are 2D images. • Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections” • In an image of one section, one must remove light from other sections • Achieving a narrow “depth-of-field” • A “confocal light microscope”

  26. Laser-Scanning Confocal Light Microscopy • Laser thru pinhole • Illuminates sample with tiny spot of light • Scan the spot over the sample • Pinhole in front of detector: Receive only light emitted from the spot

  27. Light from points that are in focus versus out of focus

  28. Spinning-disk confocal microscopy: Higher speed and sensitivity

  29. Example: Confocal imaging lessensblur from out-of-focus light

  30. Optically Sectioning a Thick Sample: Pollen Grain Multiple optical sections assembled to form a 3D image

  31. 3D Image Reconstructed From Serial Optical Sections Obtained with a Confocal Microscope

  32. Fluorescence can Measure Concentration of Ca2+ Ions in Cells: Sea Urchin egg fertilization Phase Contrast Fluorescence

  33. Summary • Light microscopy provides sufficient resolution to observe events that occur inside cells • Since light passes though water, it can be used to look at live as well as fixed material • Phase contrast and DIC optics: Good contrast • Fluorescence optics: Defined molecules can be localized within cells • “Vital” fluorescent stains: Watch particular molecular species in live cells

  34. End

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