Part 1 Principles
1. Fluorescence microscope
2. Filterset in FL-Mic
3. How concocal differs?
. What is confocal?
5. Resolution in confocal
6. Optical sectioning
7. Confocal image formation
    and time resolution
8. SNR in confocal
9. Variations of confocal

10. Special features from
     Leica sp2 confocal

Part 2 Application
1. Introduction
2. Tomographic view
    (Microscopical CT)

3. Three-D reconstruction
4. Thick specimen
5. Physiological study
Fluorescence detecting
       General consideration
Multi-channel detecting
       Background  correction
       Cross-talk correction
            Cross excitation
            Cross emission
            Unwanted FRET

Part 3 Operation and

 1. Getting started
 2. Settings in detail
     Laser line selection
      Laser intensity and 
         AOTF control

      Beam splitter
      PMT gain and offset 
      Scan speed
      Scan format, Zoom
        and Resolution

     Frame average, and
         Frame accumulation
     Pinhole and Z-resolution
     Emission collecting rang
        and Sequential scan

When Do you need confocal?
Are you abusing confocal?

Confocal Microscopy tutorial

Part 2 application of confocal microscopy

6. fluorescence detecting in confocal microscopy

        Cross-talking 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, etc..

The image here is taken from fluorescent beads which is conjugated with both FITC and TRITC.

The 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 emission.

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.

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This page was last updated 23.03.2004