Fig. 2: Lifetime characterization of the miRFP photoconversion.
a The lifetime for the ground (gray) and photoconverted (blue) forms of different miRFPs is reported (N = 3–5 repetitions, boxes mark the IQR, line defines the median point, gray dot marks the mean, whiskers mark the maximum and minimum values). The photoconversion is triggered by 405 nm illumination at 4.8 J/cm2. b Representative fluorescence lifetime decay for the far-red form (620–650 nm detection band, red dots) and NIR form (700–730 nm detection band, blue dots) of miRFP720 expressed in E. coli, together with the instrument response function (IRF) of the system (green line). c Spectral lifetime exploration for miRFP720 labeling of H2B in HeLa cells. For a non-photoconverted and photoconverted (405 nm light at 5 J/cm2) nucleus, a fluorescence lifetime image has been recorded spectrally from 640 to 740 nm, integrating intervals of 20 nm (excitation at 594 nm). The phasor plot reports the information at all the spectral window (after 8 pixels binning of the images). The two extremes of the orange dotted line correspond to lifetimes of 0.6 ns and 1.5 ns. The arrow follows the progression of the phasor in relation to the spectral progression. d Phasor plot for a layer of bacteria photoconverted only in a central square through 405 nm light in an emission region (e) where the photoconverted and non-photoconverted forms of miRFP720 overlap and therefore are indistinguishable by fluorescence intensity (680–700 nm). The two components identifiable in the phasor plot are highlighted in blue (slow component, photoconverted form) and orange (fast component, non-photoconverted form). Scale bar, 5 µm. f The same dataset has been decomposed according to the two identified lifetime components and the relative change of fluorescence is reported for the fast (0.65 ns) and slow component (1.1 ns). g Quantification of the change in fluorescence for the blue-shifted (filled bars, left) and red-shifted channel (hollow bars, right) (mean ± SD for n = 6 independent repetitions). Scale bars, Source data are provided as a Source Data file.
