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  1. 1. 1.22.2010 – Microscopy<br /><ul><li>Thicker samples than microscopy can visualize accurately.
  2. 2. Section thick tissues with a microtome.
  3. 3. Often samples are fixed/dead
  4. 4. One can use techniques of light microscopy or e- microscopy to image samples.
  5. 5. Organotypic slices are live tissues that can be put into cell cultures and analyzed. This is an exception.
  6. 6. Electric signals analyzed from this tissue
  7. 7. Confocal live imaging uses layers to excite fluorescent molecules within distinct optical layers of that tissue.
  8. 8. Three principles of microscopy
  9. 9. Contrast – requires light pass through a sample. Often cells are too clear.
  10. 10. Add dyes, or contrast, to the cell to enhance features. Works well in light and fluorescent microscopy, but often this requires the cells to be fixed and will not be able to observe movement/behavior or cells to the signals.
  11. 11. Light microscope
  12. 12. Objective lens, near to specimen.
  13. 13. Condenser lens focuses light below onto the sample of interest.
  14. 14. Resolution – a factor that measures how close two points can be in a sample and still be distinct.
  15. 15. R= (0.61*lamba)/(n * sin(angular aperture))
  16. 16. N = refractive index. Oil is about 1.5 and the larger it is, the better the resolution. The SMALLER the resolution, the closer two points can be a still be distinct structures.
  17. 17. Bestlambda is 450, oil 1.5, and angular is 70 degrees. = 194 nm. This means that two objects that are 200nm apart can be seen as distinct objects. If they are closer than this, they will not be seen as distinct structures.
  18. 18. Lambda – wavelength. This sets the limit on how an object can be and be seen by particular microscope. Smaller the wavelength, the smaller the object that can be seen.
  19. 19. E- microscopes have better resolutions.
  20. 20. Uses an electron gun as its source of light, or energy which pass through a condesnerser lens, onto specimen and
  21. 21. Has a lambda of .004nm. increase in resolution generates a maximum resolution in theory of .2nm.
  22. 22. Must be stained and fixed – gold is a good stained.
  23. 23. Use of radiation can be a drawback
  24. 24. Must be in a vacuum.
  25. 25. Magnification – how large you can make an image and still distinguish independent points. LM is 1400X in an oil. In air = 1000X
  26. 26. E- have 100X better of light microscope, so 100,000X
  27. 27. Different types of light microscopy
  28. 28. Cell often appears transparent when there isn’t manipulation
  29. 29. Brightfield stained
  30. 30. Contrast for organelles – must be dead
  31. 31. Brightfield unstained
  32. 32. Fluorescent
  33. 33. Stains used but can be alive
  34. 34. Tagged proteins for unduced expression
  35. 35. DIC – differential interference contrast imaging
  36. 36. Insertion of nomarski optic - A prism that splits light into two separate waves through specimen. Changes wavelength of light and the interference leads to increased contrast, largest difference is the edges of cells and organelles.
  37. 37. Phase contrast
  38. 38. Differentiation of organells and differences between them w/out fixing
  39. 39. Good way to see edges of cell
  40. 40. Vesicle transport in cells
  41. 41. Takes advantage of physics of light and the characteristics of it as a waveform. When two waves are in phase, they are at the same frequ and combine to create a better resolution. If out of phase, reduced intensity of light. When inphase, the unstained cell will disrupt one of the light waves creating a slowed speed for the wave form.
  42. 42. Confocal Imaging
  43. 43. Thicker samples and image optical layers for 3d images.
  44. 44. TEM vs SEM
  45. 45. 2d vs 3d
  46. 46. fluroescence microscopy
  47. 47. lots of dyes to look at parts of cells at the same times.
  48. 48. Only limited by number of filters one can place in microscope.
  49. 49. Souce of light is ultraviolet passed through an excitation filter, which removes wavelengths of light not useful in expressing the fluorescence.
  50. 50. Dyes are chemically derived that bind carbs, lipids, DNA.
  51. 51. One doesn’t need to use antibodies if the dyes bind directly.
  52. 52. Proteins most often identified by fluorescently linked antibodies because few dyes directly link to proteins.
  53. 53. Primarily, the fluorescene microscope does not have a condenser like in a light microcope. Must be thin samples.
  54. 54. confocal
  55. 55. has allowed us to use laser light to image specific layers/regions within a thick sample.
  56. 56. Images individual parts and put together to rebuild the picture.