Part 1 Principles
1. Fluorescence microscope
3. How concocal differs?
What is confocal?
Resolution in confocal
7. Confocal image formation
8. SNR in
Variations of confocal
10. Special features from
Leica sp2 confocal
3. Three-D reconstruction
4. Thick specimen
5. Physiological study
3 Operation and
2. Settings in detail
Laser intensity and
PMT gain and offset
Scan format, Zoom
Frame average, and
Pinhole and Z-resolution
Emission collecting rang
and Sequential scan
you need confocal?
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
6. fluorescence detecting in confocal microscopy
4: unwanted FRET
FRET (Fluorescence Resonance Energy Transfer) is a
phenomena encountered when emission spectrum of one fluorophore falls into excitation
spectrum of another fluorophore in the vicinity, when the two fluorophores are
close enough, usually within 1-10 nm range, then the energy from emission of the
first fluorophore will transfer to the second fluorophore and acts as excitation
energy. The final effects will be a reduction of the first emission intensity
and increase of the second emission intensity. There are many pairs of
fluorophores with this property, naming a few: like FITC and TRITC, YFP and GFP,
|The image here is taken from fluorescent beads which is
conjugated with both FITC and TRITC.
beads "should" show homogeneous green and red in the respective channel and
yellow in the overlay image because the two fluorophores are everywhere.
But here are some interesting things: In green channel, there are some areas
lacking of green on some beads, and in overlay image there are some area
showing red only. Compare these area carefully, you will find it is the area
lacking green (on the center and periphery of some beads) that shows red.
What happens? FRET. The energy of green emission has been transferred to the
red fluorophore and excites red emission, leads to the reduction or loss of
green itself, increase of red in the same area where no yellow co-localization
can be seen but
simply red emission.
So, the unwanted FRET causes false negative co-localization and a false low
intensity of the first emission. But it is not harmful for the second
The unwanted FRET is hard to correct unless you reduce the intensity of
first emission or choose other pair of fluorophore.
As discussed above, unnecessarily mixing more fluorophores in the specimen
can cause many adverse side-effects.
other tutorial |
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