MBio - Microscopy

  1. Magnification
    How much larger an object appears, compared to it's real size
  2. Resolving power/Resolution
    Minimum separation at which two points can be distinguished as separate
  3. Equation for determining resolving power
    d (Resolving power) = 0.5λ/(Numerical Aperture)
  4. Which power of ten is pico-
  5. Maximum useful magnification and resolution of light microscopy
    • 1000-2000X
    • 0.2μm (Large organelles and bacteria)
  6. Purpose of staining
    Provide contrast, increase visibility, accentuate features, preservation
  7. Fixation
    • Preserving the internal and external structures of cells (and keeping them in place)
    • Denatures enzymes and toughens structuers
  8. Types of fixation
    Heat fixation, Chemical (Formaldehyde, alcohols i.e methanol etc)
  9. When is chemical fixation used?
    • On larger, more complicated cells
    • Eukaryotes
  10. Two parts of a staining dye
    • Chromophore - Conjugated double bonds provide colour
    • Binding - Bind to cell components; Ionic, covalent or hydrophobic
  11. Difference between positive and negative staining
    • Positive; Stains the cell directly
    • Negative; Stains the background
  12. Groups of Ionic dyes
    • Basic - Pos Chomophore. E.g Crystal violet, Safranin, Malachite green and Methylene blue
    • Acidic - Neg Chromophore. E.g Eosin, Acid Fuchsin
  13. Developer of the Gram Stain
    Christian Gram (1884)
  14. Types of staining
    Gram Stain, Endospore Stain
  15. Microscopy methods requiring staining
    Genreally, only brightfield
  16. Principle of phase contrast microscopy
    Difference in refractive indices alter light intensities
  17. Uses of phase contrast microscopy
    • No staining needed
    • Studying motility
    • Studying living cell characteristics (shape)
    • Studying internal structures (endospores, inclusion bodies)
  18. How does Darkfield microscopy work (simply speaking)
    Only light that is passed through the sample is observed. The background remains black/dark
  19. Uses of Darkfield
    • Unstained cells
    • Allows alteration of lighting to achieve better imaging
    • Internal structures of large eukaryotes and motility
  20. What does DIC microscopy stand for?
    • Differetial interference microscopy
    • AKA. Nomarsky
  21. How does DIC microscopy work? (Simply)
    • Uses polarized light to detect different refractive indices and thickness
    • Brightly coloures and 3D-ish images
    • Similar to phase contrast
  22. Uses of DIC microscopy
    Ornate cell walls, endospores, granules, vacuoles, nuclei (eukaryotes)
  23. How does fluorescence microscopy work? (Simply)
    • Single wavelength of UV excites molecules in the specimen
    • Re-emitted, lower energy light is obseved
    • Often uses fluorochromes
  24. DAPI
    • 4' 6-diamino-2-phenylindole
    • Fluorochrome
    • Antibodies and DNA hybridization
  25. CSLM
    Confocal scanning laser microscopy
  26. How does CSLM work? (Simply)
    • Laser illuminates small part of specimen
    • Pinhole collects light from v. small region
    • Reduces interference problems of larger lenses
    • Sharp images
  27. Advantages of CSLM
    • Improves resolution to 1um
    • Can measure distributions and concentrations of molecules
  28. Two photon microscopy
    • Uses two red-light photons
    • Greater penetration
  29. Resolution and magnification of Electron Microscopy
    • 0.2nm
    • 100,000X
  30. Transmission Electron Microscopy
    • Low penetration
    • Study cell ultrastructure
    • Preparation is lethal to specimens
  31. TEM Preparation
    • Chemical Fixation
    • Physical prep. - Thin sectioning
    • Staining (Positive, Negative and Staining) - Osmic acid, permanganate, uranium salts
  32. Scanning electron microscopy
    • Uses electron scattering
    • Specimen coated in heavy metal
    • 3D Image/Depth of field
    • Resolution 7nm/15X - 10,000X
  33. Scanned probe microscopy
    • Sharp probe used to poke surface
    • Often uses single atom at tip
    • Sample prep not needed
    • 1,000,000,000X Magnification
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MBio - Microscopy