Table 17.9.1.

Troubleshooting Guide for Acceptor-Photobleaching FRET

Problem	Possible cause	Solution
FRET efficiency low, even for positive control	Acceptor fluorophore photobleached before performing FRET measurement	Do not image labeled cells with a fluorescent lamp, which will cause significant photobleaching; instead use low intensity laser power, using the donor fluorescence to find cells and focus them. Keep imaging time to a minimum before making the FRET measurement. Any loss of acceptor fluorescence will reduce the difference between the quenched and unquenched donor fluorophore.
	Acceptor fluorophore incompletely photobleached	Be sure to identify photobleaching conditions to reduce fluorescence of the acceptor to background levels; otherwise, the donor will not be completely dequenched.
High and low FRET observed along edges of fluorescent structures	Pre- and post-bleach images not registered, causing a slight shift of positions of areas with low fluorescence in a pre-bleach image to areas with significant fluorescence and resulting in an apparent increase in the donor channel	Align pre- and post-bleach images in an image manipulation program (e.g., Photoshop, ImageJ or NIH Image; then perform FRET analysis with the modified images. Note that the authors' macro includes a registration step.
FRET detected when using fluorescent fusion proteins as negative controls. (if the proteins used authentically do not interact)	Fused fluorescent proteins dimerizing (may occur at high concentrations; see Zacharias et al., 2002 and Snapp et al., 2003)	Be sure to make fluorescent fusion proteins with fluorescent proteins that contain monomerizing mutations.
FRET observed between negative control proteins in a fluorescence intensity-dependent manner	FRET due to density-dependent clustering of proteins most likely caused by (1) nonspecificity of an antibody or (2) labeled proteins are present at such high concentrations that their proximities are sufficiently close for FRET to occur	Confirm specificity by preincubating antibody with peptide that antibody was raised against; restrict analysis to regions of cells in which FRET occurs in an intensity independent manner (problem may be unavoidable in cases where the protein is concentrated within an organelle); use smaller fluorescent probes instead of antibodies; or consider other FRET methods including FLIM and fluorescence anisotropy (see supplementary data and appendix in Kenworthy and Edidin, 1998 and Sharma et al., 2004).