Fig. 2: Unmixing of two fluorescent proteins in the same pixel.

a Live cells (middle panels) expressing tandem mTFP1-linker-mTurquoise2 (mTFP1-mTQ2), mTFP1-T2A-mTQ2 which expresses equimolar amounts of both fluorescent proteins separately and co-expression of mTFP1 and mTQ2 with transient transfection were tested to unmix both populations of plasmids. The lifetime histograms in nanoseconds (ns) corresponding to each fluorescent protein expressed separately (dark gray) and together (light gray) for each case is also shown, the lifetime histograms present a color bar that goes from 0 (violet) to 5 ns (green). Scale bar 5 μm. Bar charts (right panels) plotting the lifetimes of mTFP1 (dark blue, 2.8 ns) and mTQ2 (cyan, 4.12 ns) when expressed alone were employed as a reference as explained in methods. The percentage obtained for the three cases depicted were 53 ± 4%, n = 8 (tandem mTFP1-mTQ2); 53 ± 7, n = 10 (mTFP1-T2A-mTQ2) and 53.5 ± 4%, n = 8 (mTFP1 + mTQ2) where n is the number of cells examined over 3 independent experiments; respectively. b The same cells shown in (a) are here analyzed with the average lifetime non-fitting approach. Micrographs show cells expressing mTFP1-mTQ2 (first row), mTFP1-T2A-mTQ2 (second row) and mTFP1 + mTQ2 third row. The intensity bars in the micrographs denote: number of photons (first column). The pixel-by-pixel fraction of the corresponding fluorophore and corresponding histograms are also shown for comparison and validation with the fitting approach (middle and right columns). Scale bar 2 μm. The average proportions where 48.5 ± 5%, n = 10 (tandem mTFP1-mTQ2); 57.3 ± 6, n = 10 (mTFP1-T2A-mTQ2) and 47.8 ± 7%, n = 10 (mTFP1 + mTQ2) where n is the number of cells examined over 3 independent experiments. c The Phasor Plot was employed with the same cells and similar results were obtained, both in terms of lifetime and proportions, for mTFP1 and mTQ2. Both phasor signatures for mTFP1 and mTQ2 fall in the universal circle indicating that these two fluorescent proteins behave as single exponentials. The bar charts show the proportions for mTFP1 where 47 ± 3%, n = 10 (tandem mTFP1-mTQ2); 57.4 ± 8, n = 10 (mTFP1-T2A-mTQ2) and 46.5 ± 9%, n = 10 (mTFP1 + mTQ2) where n is the number of cells examined over 3 independent experiments. Data are presented as mean values +/- SD” as appropriate. Source data are provided as a Source Data file.