FRET Measurements by TCSPC FLIM

FRET measurements are an established teehnique used to determine distances in cells on the 1-nm scale. The general principle of FRET [169, 230, 308] is shown in Fig. 5.4, page 64. The fluorescence emission band of a donor molecule overlaps the absorption band of an acceptor molecule. If both molecules are in close interaction, a radiationless energy transfer from the donor to the acceptor occurs. The efficiency of the energy transfer increases with the 6th order of the reciprocal distance. 

The obvious difficulty of FRET measurements in cells is that the concentrations of the donor and acceptor molecules are variable and unknown. Moreover, the emission band of the donor extends into the emission band of the acceptor, and the absorption band of the acceptor extends into the absorption band of the donor. A number of different FRET techniques address these implications. 

Steady-state FRET imaging uses the ratio of the donor and acceptor fluorescence intensities as an indicator of FRET [403]. The problem of the ratio technique is that the concentrations of the donor and acceptor may vary independently, resulting in unpredictable errors. 

The influence of the concentration can be largely avoided by calibrating the crosstalk of the donor fluorescence in the acceptor detection channel and the amount of directly excited acceptor fluorescence [159, 360, 402, 535]. The calibration employs different cells, each containing only the acceptor and the donor, 

It is commonly accepted that the most reliable way to measure FRET in cells is the acceptor-photobleaching technique. A donor image is recorded, then the acceptor is destroyed by photobleaching, and another donor image is recorded. The FRET efficiency is obtained from the relative increase of the donor fluorescence intensity [205]. The drawback of the technique is that it is destructive. It is therefore impossible to run successive FRET measurements in the same cell. It is also difficult to use in living cells because the acceptor recovers after photobleaching by diffusion effects. 

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