Microscopes: LM (Phase and Fluorescence)

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Microscopes: LM (Phase and Fluorescence)
2013-09-01 18:54:10
Cell Bio

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  1. True or False
    small, unstained specimens are hard to see with bright-field microscopes
  2. phase- contrast microscope
                                                                  i.      makes highly transparent objects more visible

    •  1.      parts of an object affect light differently from one another= easy to analyze parts
  3. Why do parts of an object affect light differently?
    •                                                                                                                                       i.      refractive index: cell organelles have different proportions of various molecules, which have different indeces
    • 1.      not seen by eye, but the PCM converts differences in refractive indeces into differences in intensity
  4. How does the PCM convert refractive indeces into differences in intensity?
    • a.      separating the direct light that enters the objective lens from the diffracted light emanating from the specimen
    • b.      causing light rays from the two sources to interfere with one another
  5. Phase Contrast is most useful for __. 
    Has optical handicaps that result in __.
    •                                                               i.      examining intracellular components of living cellsat high resolution
    • result in loss of resolution (image suffers from interfering halos and shading produced where sharp changes in RI occur
  6.                                                               i.      It is an __; others minimize problems by completely separating __

    1.      another is __): delivers __quality image
    a.      contrast relies on __ 

    b.      edges of structures= __
    • interference microscope
    • direct and diffracted beams using light paths and prisms
    • differential interference contrast (Nomarski interference
    • 3D 
    • rate of change of RI across specimen
    • great contrast
  7. a.      Fluorescence Microscopy (and Related Techniques)
                                                                  i.      allows viewers to see locations of certain molecules __
    • fluorophores/ fluorochromes)
    • absorb invisible, UV radiation, and release a protion of the energy in the longer, visible wavelengths (fluorescence
  8. In FM, the light source produces a beam of __ that travels through a __, which blocks all __except the one that excites __.

    1. this beam is focused on the specimen containing the __, which becomes excited and __ that is focused by the __ into an image seen by viewer
    • UV light
    • filter
    • wavelengths 
    • fluorophome
    • fluorophore
    • emits light of a visible wavelength
    • objective lens
  9. 1.      because the light source produces only __, objects stained with __appear __against a black background= very high contrast
    • UV (black) light
    • fluorophore 
    • brightly colored
  10. Applications of FM:

    fluorescent antibodies (immunofluorescence), locating (like DNA with specific nucleotide sequences), sizing of molecules that can fit between cells, or as probes
  11. 1.      biomolecules were made fluorescent by conjugating them with a synthetic organic __; there are also naturally fluorescent molecules
  12. a.      __ discovered certain jellyfish have fluorescent proteins (aequorin, GFP, etc.)
    Osamu Shimomoura
  13. a.      __, etc. cloned gene that encodes __ and put in another organism
                                                                                                                                          i.      led to use of __to study __ of proteins in living cells
    __doesn’t need __ to __ 
    Why not?
    • Douglas Prasher, Martin Chalfie
    • GFP
    • GFP 
    • spatial/ temporal distribution
    • GFP 

    • cofactor
    • absorb and emit light
    • self-modification of three of the amino acids that make up the primary structure of GFP
  14. a.      use of __to link GFP with other gene to follow it
                                                                                                                                          i.      __ studies can use __variants that have different shades generated by __ through directed __ of the GFP gene 

    __ isolated from sea anemone
    • recombination 
    • live cell imaging
    • GFP 

    • Robert Tsien
    • mutagenesis
    • red fluorescent tetrameric protein
  15.                                                               i.      GFP variants also useful in __, which can measure distances between fluorophores in the nanoscale range
    fluorescence resonance energy transfer (FRET
  16. FRET can __.
    This transfer of energy reduces the __ of hte donor and increases the __ of the acceptor.
    1.      measure changes in distances between two protein parts or separate proteins (in vitro or w/in living cell)based on fact that excitation energy can be transferred from one fluorescent group to another, as long as they are really close

    • fluorescence intensity
    • fluorescence intensity
  17. In FRET, efficiency of transfer __.
    Due to this, __ during a cell process provides measure of changes in distance between them at different stages
    • decreases with increasing distance
    • determination of changes
  18. FRET can also be used to __.
    follow protein folding or membrane associations
  19.                                                               i.      += that examine images and score the characteristics was created
    1.      good for __ of cells that were subjected to __, looking for genes encoding proteins that are involved in certain cell processes, etc
    • computational programs
    • screening phenotypes
    • siRNAs
  20.                                                               i.      __: fluorophores present within cells are excited by the simultaneous arrival of two or more __

    1.      the longer the wavelength, the __
    a.      can help locate deep proteins
    • multiphoton microscopy
    • photons of longer wavelength
    • less its energy and the greater its penetrating power