Separation of FRET from autofluorescence and background noise is illustrated
Top: Spectrally resolved fluorescence images (at six selected wavelengths out of a total of 200 collected) obtained from yeast cells expressing two populations of IRE1, one of which is fused to GFP2 and the other to YFP, respectively, both residing at specific loci in the endoplasmic reticulum.
Bottom: A multi-component spectral decomposition method was used to separate autofluorescence (AF), donor fluorescence in the presence of acceptor (kDA, GFP2), and acceptor fluorescence in the presence of donor (kAD, YFP) from pure noise (visible both inside and outside the cells). An apparent FRET efficiency image (EApp) was computed on a pixel-by-pixel level from kDA and kAD. Notice that the noise was removed from both the endogenous (i.e., autofluorescence) and exogenous fluorescence (from the fusion proteins) images. Arrows in the last three figures indicate the position of distinct IRE1 complexes (that are at least dimeric in size).
More details are given in Mannan et al, Journal of Molecular Biology, Forthcoming 2013. Images, courtesy of Dey lab, UW-Milwaukee Department of Biological Sciences (samples) and Raicu lab UW-Milwaukee Physics Department (imaging and analysis).