Microscopes Part One

  1. Why are cells so hard to study?
    WHat do we do?
    • they are so tiny; most are colorless
    • use microscopes
  2. Robert HOoke (1660s) 
    __= when microscopes became available
    discovered __
    1) What did he see? What did he come up with?
    • 1600s
    • cell walls of cork (plant)
    • holes and cell walls
    • came up with word cell after monks who live in monasteries
  3. Anto von Leeuwenhoek
    a) __/ great __
    b) First to see what?
    • a) smaller microscope/ great lensmaker (could magnify up to 200x)
    • b) single-celled microorganisms
  4. Light Microscope
    past versus present
    preps and stains are pretty similar
  5. If a microscope uses __, a limiting factor is __
    • radiation
    • resolution
  6. what is resolution?
    minimum distance two objects can be and still be distinguished as two different objects
  7. What is the case if something is smaller than resolution?
    you won't see it and magnificaiton can't bring it back
  8. True or False:
    The millimeter is greatly used in Cellular Biology.
    • False:
    • it is not
  9. Types of resolution
    • eye: 0.1 mm
    • most cells: 1/10 of a mm in diameter
    • light microscope: 0.0023 mm= 0.25 um= 250 nm= 2500 Angstroms
  10. What determines resolution?
    • formula:
    • d= (0.61λ)/ (nsinα)
  11. Explain what everything means in the formula.
    • d= resolution
    • λ= wavelength of radiation
    • n= refractive index (air= 1/ oil= 1.5)
    • α= half of the angle of the cone of light going into the lens
    • the whole denominator is the numerical aperture
  12. What is RI?
    the velocity of light in the vacuum divided by the velocity of light or radiation in transmitting medium between specimen and objective lens
  13. Which is better, a small value for d or a large value for d. Why?
    smaller because the larger the resolution is, the less you can see. The smaller it is, the smaller the things can be.
  14. How can the resolution be improved?
    • make the half angle larger
    • make the wavelength shorter
    • increase the refractive index
  15. What is the maximum half angle?
    seventy degrees
  16. max sinα is?
  17. What can you see with a LM?
    • nucleus (maybe the nucleolus)
    • cell wall
    • chloroplasts (but not internal structure)
  18. What can't you see with a LM?
    • organelles
    • cell membrane
  19. The light microscope is __.
  20. Magnification
    The limit of resolution does what to magnification?
    sets upper limit for what useful magnificaiton is going to be
  21. The greatest useful magnification that can be used is about __ .
    1000 x the numerical aperture
  22. With a LM, most cells are __. Therefore, we do what?
    stain the specimen
  23. With stains, what can you see?
    where things are, not see more things
  24. Stains don't __ at all.
    improve resolution
  25. Three problems of stain use.
    • They lead to dead tissue:
    • 1) some stains are toxic and can't be used with living cells
    • 2) conditions needed to get the stain into cell are toxic
    • 3) stain simply doesn't penetrate cell unless the cell is dead
  26. Stains are useful for different purposes depending on __. If you choose the right stain, you can find a __. Explain that.
    • what you want to localize
    • particular enzyme
    • 1) must find substrate for that; and, substrate wouldn't have color; product would
  27. Modifications in LM
    1) Fluorescene Microscopy
    What is it?
    if a compound is fluorescent (shine light on it), it absorbs wavelength--> reemitted in wavelength of longer length (sometimes, heat is released as well)
  28. Fluorescence Microscopy
    1) one or more __ in compound absorbs __. Explain what it does to electrons.
    • electrons
    • specific wavelength
    • electrons are excited and made unstable
  29. In FM, light emitted is always at __.
    Radiation used with fluorescence is __ or __
    • a longer wavelength
    • UV or blue
  30. Fluorescent Compounds
    • naturally occurring things that will naturally fluoresce (chlorophyll, collagen, etc)
    • if a compound doesn't fluoresce naturally, you can change the structure to allow it to fluoresce
  31. Fluorescent __ and __ can also be added. Will bind to a particular molecule.
    • probes
    • dyes
  32. Advantage of FM.
    How common ?
    • you can use them with living cells
    • not common, since most are not specific to what they bind to
  33. Immunofluorescent antibody staining/ labeling= __
    - __ attached to compound that specifically attaches to other molecules
    - advantage: __. 
    - attaching a dye to antibody to another does what?
    • immunofluorescence
    • nonspecific probe
    • very specific
    • concentrates it a bit
  34. Green Fluorescent Protein
    - isolated from __. 
    - small, natural, occurring in jellyfish and naturally __.
    • jellyfish
    • fluoresces
  35. With GFP, you can use __ to __. Every time its being used or transcribed, it will __. 
    Shine a UV Light--> __. 
    You can use one or many fluorescent probes.
    • recombination
    • attach it to a gene in another organism to follow the desired protein
    • fluoresce
    • green color --> able to follow
  36. Lasers (Confocal)
    for a sharper image of the specimen
  37. - one of the problems of putting tissue through long prep is what?
    you never know if what you're looking at is the actual way it looked when you began
Card Set
Microscopes Part One