Experimental FLIM measurements on fluorescent solutions with our fast-FLIM prototype.

Lifetime images of Rd6G alone, AO alone and of three mixtures of Rd6G and AO with theoretical fractions of interacting donor of 0.17, 0.44 and 0.65 were acquired with N≈1500 photons. The polar plot and the mean lifetime images of each solution are represented in (A). All spots corresponding to the mixtures in the polar plot are well localized on a line connecting pure Rh6G and pure AO. We have also calculated fDP, fDM and mfD for the three mixtures and the corresponding images are indicated in (B). We show in (C) the images of donor lifetime in presence of the acceptor estimated with the polar approach and the moment method. We have also performed FLIM acquisitions with different numbers of detected photons for each fraction of interacting donor: 0.17 (D), 0.44 (E) and 0.65 (F); the corresponding plots of fDP, fDM, mfD, τFP and τFM as a function of N are reported in the right part. For all graphs, markers with error bars represent the medians and interquartile ranges of FLIM images. The theoretical fD and τF are indicated in dotted lines.