Centre Optical Instrumentation Laboratory

Acceptor Photo-Bleaching FRET

Photo bleaching the acceptor fluorophore has the effect of dequenching the donor fluorescence, resulting in an increase in the donor fluorescence. This method has the advantages of being easy to implement requiring few controls and very little image processing.

Photobleaching equation

Bleach Equation

Although controls are not always used it is good practise to use single labelled donor and acceptor specimens to check the uniformity of staining or transfections and that co-transfecting/dual labelling hasn't caused an alteration in the expression of your protein or labelling of your antibody. It is also possible that high concentrations of one or other fluorophore can produce self quenching which will have an adverse effect on your FRET efficiency.


Acceptor Only Labelled Specimen

1. Collect an image using settings for the acceptor fluorophore only
2. This is technically not really neccessary but its a good idea to check your acceptor labelling/expression levels to ensure that you are actually bleaching something other than bleed through

Dual Labelled FRET specimen

1. Collect a pre-bleach image using settings for the donor fluorophore only
2. Collect a pre-bleach image using settings for the acceptor fluorophore only (used to check the presence of acceptor labelling)
3. Using the donor settings remove any neutral density filters (widefield systems) or turn the acceptor laser power to maximum (confocal systems)
4. Illuminate the acceptor until it has completely bleached (the time will depend on the power of the illumination and the fluorophore)

See images below for an example taken from a positive control

Donor Pre-Bleach

Donor Post Bleach

Donor Pre

Donor Postbleach

Acceptor Pre-Bleach

Acceptor Post-Bleach

Acceptor Prebleach

Acceptor Post Bleach

Positive control: HEK cells (line 293T)
Donor = eGFP from Clontech peGFP-C2 vector
Acceptor = mRFP, NCBT Ref = AF506027 (See Campbell et al PNAS 2002)

Calculation (measured from the bright area in the lower right area of the cell)

These calculations wer performed using Metamorph, although most image analysis packages are capable of measuring FRET using this technique.

All values are grey values


(141.17-1.1) - (78.05 - 1.0)

(141.17 - 1.1)




This method does not correct for any spectral bleed through problems and does not correct for any variations in fluorophore concentration. For this reason it is best used as guide/check for sensitized FRET

Supporting References

Campbell et al PNAS, 2002 Jun 11;99(12):7877-82.

Erickson et al, Neuron 2001; 31: 973-985