Figure 3.

Proof of principle. (a) Shown at the top is a schematic illustration of the tandem fluorescent protein fusion, with N- and C-terminally tagged spacer protein (Don1). Shown at the bottom, filtered auto- and cross-correlation functions were calculated for selected combinations of FPs. Fluorescence proteins in rows (blue lines) were attached N-terminally and proteins in columns C-terminally (red lines). Black lines correspond to the cross-correlation curves. Insets show corresponding fluorescence lifetime histograms (x axis is number of channels ranging from 0 to 2000 and y axis equate to normalized number of events ranging from 10⁻⁴ to 10°). (b) Comparison of the sc-FLCCS analysis for selected pairs of FPs as determined by R²-values. The higher the R²-value, the better the sc-FLCCS filtering and consecutive analysis. The difference in average fluorescence lifetimes between FPs (τ_diff) is listed in the upper right corner of the corresponding box plot. (c) Comparison of the positive (left) and the negative (right) controls is shown. At the top is schematic illustrations of the strains. Orange lines denote weak CYC1 promoter. In the middle, cross-correlation curves, where solid dark line corresponds to the mean curve, light borders, denote standard deviations. At the bottom are the overall concentrations resulted from autocorrelation curves for each diffusing species and their complexes as determined by the sc-FLCCS analysis. Dashed line separates individual strains.