Fig. 7: Signal separation in pairwise FLIM multiplexing of intracellularly targeted FAST variants in live HeLa cells.

The HBR-2,5-DM fluorogen was used in all cases. The fitting-based approach with fixed τ from previously obtained data for corresponding complexes (see Methods for more detail) was applied to separate time-resolved fluorescence signals from differently localized FAST probes, including co-localized signals. a Color-coding legend. The lifetime values shown in the legend were obtained via the monoexponential fitting of FAST-R52K, P68T, and F62L expressed as H2B-fuses (Fig. 3a, Supplementary Table S9). Color brightness indicates the relative contribution (amplitude) of the respective exponential component calculated via the full-image biexponential fitting using the fixed lifetime values mentioned above. b–e Live-cell fluorescence lifetime microscopy of HBR-2,5-DM fluorogen in complexes with the genetically targeted FAST variants expressed in HeLa cells. Grayscale leftmost images show fluorescence intensity; composite images are designed to show the distribution (color) and amplitude (brightness) of two fluorescence populations calculated based on the full-image biexponential fitting; images named according to specific fusion partners are designed to show the distribution of individual fluorescence population represented within the image. b H2B-F62L and R52K-vimentin (representative image, similar results in n = 18 cells). c H2B-F62L and P68T-vimentin (representative image, similar results in n = 17 cells). d H2B-R52K and P68T-vimentin (representative image, similar results in n = 17 cells). e H2B-F62L and β4Gal-T1-R52K (Golgi apparatus) (representative image, similar results in n = 12). Scale bar 10 micrometers.