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Chapter 9

Chapter 9. Visualizing Cells. A sense of scale between living cells and atoms. Resolving power. The light microscope can resolve details 0.2 m m apart. The limit of resolution of a light microscope is set by the wavelength of visible light from

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Chapter 9

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  1. Chapter 9 Visualizing Cells

  2. A sense of scale between living cells and atoms

  3. Resolving power

  4. The light microscope can resolve details 0.2 mm apart The limit of resolution of a light microscope is set by the wavelength of visible light from about 0.4 mm (for violet) to 0.7 mm (for deep red). Under the best conditions, with violet light and a numerical aperture of 1.4, a limit of resolution of 0.2 mm can be obtained.

  5. The wave nature of light causes optical diffraction effects When two light waves are in phase, the amplitude of the resultant wave is larger and the brightness is increased. When two light waves are out of phase, they cancel each other partly and produce a wave whose amplitude, and therefore brightness, is decreased.

  6. Edge effects The interaction of light with an object changes the phase relationships of the light waves producing complex interference effects. For example, at high magnification, the shadow of a straight edge that is evenly illuminated appears as a set of parallel lines, whereas that of a circular spot appears as a set of concentric rings.

  7. Two ways to obtain contrast in light microscopy (A) The stained portion of the cell will absorb light of some wavelengths, which depend on the stain, but will allow other wavelengths to pass through it. A colored image of the cell is obtained that is visible in the normal bright-field microscope. (B) Light passing through an unstained cell undergoes very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. The phase of the light, however, is altered by its passage through either thicker or denser parts of the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope.

  8. Four types of light microscopy (A) Bright-field microscopy (B) Phase-contrast microscopy (C) Nomarski differential-interference-contrast microscopy (D) Dark-field microscopy

  9. Images can be enhanced and analyzed by electronic techniques

  10. Tissues are usually fixed and sectioned for microscopy

  11. Different components of the cell can be selectively stained

  12. Sectioned tissue can be used to visualize specific patterns of differential gene expression RNA in situ hybridization

  13. Specific molecules can be located in cells by fluorescence microscopy

  14. Fluorescent probes

  15. Multiple-fluorescent-probe microscopy

  16. Antibodies can be used to detect specific molecules Immunofluorescence

  17. Indirect immunocytochemistry

  18. Imaging of complex three-dimensional objects is possible with the optical microscope Image deconvolution

  19. The confocal microscope produces optical sections by excluding out-of-focus light

  20. Comparison of conventional and confocal fluorescence microscopy

  21. Fluorescent proteins can be used to tag individual proteins in living cells and organisms Green fluorescent protein (GFP) – the structure shows the eleven b strands that form the staves of a barrel. Buried within the barrel is the active chromophore that is formed post-translationally from the protruding side chains of three amino acid residues

  22. GFP-tagged proteins

  23. Protein dynamics can be followed in living cells

  24. The electron microscope resolves the fine structure of the cell Thin section of a yeast cell showing the nucleus, mitochondria, cell wall, Golgi stacks, and ribosomes in a state that is presumed to be as life-like as possible

  25. The limit of resolution of the electron microscope Individual files of gold atoms resolved by transmission electron microscope. The distance between adjacent files of gold atoms is about 0.2nm.

  26. Principal features of a light microscope and a transmission electron microscope

  27. Biological specimens require special preparation for the electron microscope Two common chemical fixatives used for electron microscopy

  28. Copper grid that supports the thin sections of a specimen in a TEM

  29. Specific macromolecules can be localized by immunogold electron microscopy

  30. Images of surfaces can be obtained by scanning electron microscopy

  31. The scanning electron microscope

  32. Metal shadowing allows surface features to be examined at high resolution by Transmission Electron Microscopy Preparation of a metal-shadowed replica of the surface of a specimen

  33. Negative staining and Cryoelectron microscopy allow macromolecules to be viewed at high resolution Negatively stained actin filaments

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