When energy transfer occurs, the donor emission will be quenched because of the direct transfer of excitation energy to the acceptor (see FRET Basics). If the acceptor fluorophore is destroyed, FRET will be eliminated and the donor signal will be de-quenched. The increase in the donor signal resulting from de-quenching is a direct measure of the efficiency of FRET. The technique of acceptor photobleaching FRET (apFRET) exploits this characteristic, and measures the de-quenching of the donor signal in the regions of the cell where FRET had occurred.

The apFRET method requires the selective bleaching of the acceptor, because any bleaching of the donor fluorophore will lead to an underestimation of the de-quenching. Further, bleaching of the acceptor should be as close to completion as possible, since any remaining acceptor will still be available for FRET, again resulting in an underestimation of the donor de-quenching. The YFP variant is sensitive to photobleaching making it useful for FRET measurements by acceptor photobleaching. The results shown above (left panel) demonstrate the selective photobleaching of YFP in cells that also co-expressed CFP. Greater than 90% bleaching of YFP was achieved with less than 5% bleaching of the co-expressed CFP under these conditions. More rapid selective bleaching can be achieved with different light sources or laser lines.

1. As described in the protocol for wide-field fluorescence, scan the field of cells to find healthy cells that are expressing reasonable levels of the acceptor-fusion protein. Using transient co-transfection of expression vectors we find good agreement between the expression levels for both donor and acceptor fusion proteins. Therefore, scanning the field for cells expressing a certain level of acceptor fluorescence is often a good predictor of the donor expression level in that cell and, importantly, avoids photobleaching of the donor.

2. Acquire and save the acceptor fluorescence image (Acc1, above). This provides a reference image for the acceptor expression level and photobleaching.

3. With the excitation shutter closed, switch to the CFP filter set and acquire the donor image (Don1, above) at the same focal plain as the acceptor reference image. Save the image for processing.

4. For photobleaching of the YFP acceptor, we have used approximately 5 minute exposure of the specimen to unfiltered 505 nm excitation light. This typically reduces the YFP fluorescence signal more than 10-fold. At completion, acquire a second acceptor reference image under identical conditions to the first acceptor image to document photobleaching.

5. Acquire a second donor image (Don2, above) under identical conditions used for the first image with the CFP filter set.

6. The images are then processed by background-subtraction. Images before and after acceptor photobleaching can be combined in a single mosaic image for the direct comparison of signal level. The digital image of the donor before acceptor photobleaching can also be subtracted from donor after bleaching to demonstrate donor dequenching, provided there has been no significant movement of the cell during the period of the experiment. An intensity profile map of the donor2-donor1 image can be used to represent the change in donor signal (above), or the images can be combined into a single mosaic image and a look-up-table can be applied to indicate the pixel-by-pixel fluorescence signal intensity in the side by side images. For direct comparison, histograms of the signal levels can be plotted for both donor 1 and donor 2 fluorescence for the entire cell or for specific regions of interest (ROI).

7. Control experiments characterizing co-localized, but non-interacting proteins, or alternatively, mutant proteins that fail to interact, are critical for demonstrating the specificity of interactions measured by FRET approaches. The levels of control protein expression should be as similar as possible to those acquired under the experimental conditions.