Fluorescence Microscopy
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FLUORESCENCE MICROSCOPY APPLICATIONS
Fluorescence recovery after photobleaching (FRAP)
- Used to study membrane fluidity
Fluorescence resonance energy transfer (FRET)
- Used to study protein-protein interactions
FLUORESCENCE MICROSCOPY METHOD
1. Light passes through excitation filter
2. Excitation filter filters out undesired wavelengths of light
3. Mirror deflects light downward toward sample
4. Light passes through the objective lens and onto specimen of interest
5. Molecules in sample absorb light & emit light with longer wavelength (fluoresce)
6. Fluorescent light travels back upward and passes through mirror w/o deflecting
7. Barrier filter above the mirror lets fluorescent light through
8. Fluorescence observed
FRAP
1. Tag membrane proteins with fluorophore (i.e. GFP)
2. Irreversibly bleach portion of membrane with laser (photobleaching)
3. Measure rate at which membrane recovers fluorescence (proportional to rate at which tagged molecules diffuse back into bleached area)
FRET
1. Tag one protein with blue GFP and another with green GFP
2. Shine violet light on sample
If the proteins interact (i.e they come in close proximity):
- Blue light from blue GFP excites green GFP
- Green light observed
If the proteins do not interact:
- Blue light observed (not absorbed and reemitted by green GFP)