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True or False
small, unstained specimens are hard to see with bright-field microscopes
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
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
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
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
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
- rate of change of RI across specimen
- great contrast
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
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
- emits light of a visible wavelength
- objective lens
1. because the light source produces only __, objects stained with __appear __against a black background= very high contrast
- UV (black) light
- brightly colored
Applications of FM:
fluorescent antibodies (immunofluorescence), locating (like DNA with specific nucleotide sequences), sizing of molecules that can fit between cells, or as probes
1. biomolecules were made fluorescent by conjugating them with a synthetic organic __; there are also naturally fluorescent molecules
a. __ discovered certain jellyfish have fluorescent proteins (aequorin, GFP, etc.)
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 __
- Douglas Prasher, Martin Chalfie
- spatial/ temporal distribution
- absorb and emit light
- self-modification of three of the amino acids that make up the primary structure of GFP
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
1. __ isolated from sea anemone
- live cell imaging
- Robert Tsien
- red fluorescent tetrameric protein
i. GFP variants also useful in __, which can measure distances between fluorophores in the nanoscale range
fluorescence resonance energy transfer (FRET
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
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
FRET can also be used to __.
follow protein folding or membrane associations
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
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