SPIM and FLIM confirm FRET in full length RPTPα-CFP and -YFP expressing cells. (A) Normalized uncorrected fluorescence emission spectra of plasma membrane regions of single SK-N-MC cells expressing both RPTPα-CFP and RPTPα-YFP (closed circles) or SK-N-MC cells expressing both RPTPα-CFP and EGFR-YFP (control, open circles) as obtained by fluorescence spectral imaging microscopy (SPIM). The emission spectra were normalized to the first emission peak of the CFP spectrum (at 475 nm) for comparative reasons (note that due to this normalization procedure no 'quenching' of CFP fluorescence due to FRET seems apparent from the figure). (B) CFP-fluorescence lifetime images (τφ, calculated from the phase shift) of cells expressing both RPTPα-CFP and RPTPα-YFP (left) or RPTPα-CFP and EGFR-YFP (control, right). The histogram represents the distribution of fluorescence lifetimes throughout the image. (C) Correlation between FLIM and SPIM data of cells expressing both RPTPα-CFP and RPTPα-YFP (closed symbols) or RPTPα-CFP and EGFR-YFP (control, open symbols). Each point represents a combined SPIM and FLIM measurement of a single cell. On the horizontal axis the normalized intensity of the emission spectrum at 527 nm is indicated (which is proportional to the YFP/CFP fluorescence emission ratio). Lifetimes determined from phase (τφ) and modulation (τM) are plotted with circles and squares, respectively. Regression analysis indicated a -85% and -88% correlation coefficient for the respective lines. Error bars represent pixel-by-pixel standard deviations in fluorescence lifetimes or fluorescence emission spectra.