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Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

Figure 6

Global analysis of a polarisation resolved homo-FRET TCSPC dataset reading out PtdIns(3,4,5)P3 accumulation at the membrane.

A MEF transfected with EGFP-AKT-PH was imaged at two minute intervals and stimulated with 50 ng/ml PDGF after 6 minutes (indicated by black triangles). (A, top row) False colour map of the initial anisotropy contribution r2 associated with homo-FRET over the time course. (A, bottom row) Integrated fluorescence intensity images over the time course. (B, C) Initial anisotropy contributions spatially averaged over the cell: r1 associated with the rotational correlation (B) and r2 associated with homo-FRET (C). Error bars represent the standard deviation across the image. (D,E) Exemplar fluorescence decays from the region indicated by a white triangle in the first (D) and last (E) frame with fit (top) and normalised residuals (bottom). The thin, fainter lines represent the experimental data while the thick, bolder lines represent the fitted model. Black lines represent the parallel component while grey lines represent the perpendicular component. Data are representative of three experiments. White scale bar represents 20 µm.

Figure 6