Figure 3.

Results obtained from spectral FRET from cells expressing the fusion proteins Ste2p-GFP₂ and Ste2p-YFP in internal and plasma membranes of yeast (S. cerevisiae) (upper row) and GFP₂-GG-YFP in the yeast cytoplasm (lower row). (a and b) Spectral images obtained from measurements with a two-photon microscope with spectral resolution were unmixed (36) to obtain the donor (k^DA) and acceptor (k^AD) signals for each pixel. (c) From the signals obtained, the pixel-level apparent FRET efficiency (E_app) was calculated according to Eq. 10. (d) The FRET efficiency map shown in c was used to generate the distribution of measured FRET efficiencies. Data points represent the experimental distributions. The data were fitted to a sum of five (upper row) or two (lower row) Gaussians shown individually (thin green lines) or as a sum (thick red line).