Fig. 3.

In vitro fluorescence lifetime measurement of GFP bound to chromatin for range of doxorubicin concentrations. a Exemplar fluorescence lifetime images of IGROV-1 cells with stable expression of histone H1-EGFP treated with a range of doxorubicin concentrations (0, 0.18, 0.9, 1.8, 9, 18 µM) for 3 h then fixed, washed and imaged with the multiphoton LSM (scale bar is 120 µm). For each concentration 3 or more fields-of-view (containing a total of >100 nuclei) were acquired. b Exemplar fluorescence lifetime images of the same samples as in a but acquired with the CEM (scale bar is 120 µm). In a and b FLIM data was fitted to a single exponential decay model (as discussed in the Methods section). c Plot shows the mean lifetime of segmented nuclei averaged over the mean fluorescence lifetimes per segmented nucleus (i.e., average of values from pixel-wise fitting to a single exponential decay model within a single nucleus) for each doxorubicin concentration; d plot shows the intensity-weighted mean H1-EGFP fluorescence lifetimes obtained by global fitting across the entire dataset (as discussed in the Methods section) to a double exponential decay model with the long lifetime component fixed to the value obtained from cells expressing H1-EGFP when not treated with doxorubicin. For c and d black and magenta lines and circles represent the multiphoton LSM and CEM data, respectively. e plot shows only the globally fitted intensity-weighted mean H1-EGFP lifetime for the experimental CEM FLIM data (magenta lines and circles) together with simulations of the measured lifetime dependence on doxorubicin concentration (as discussed in Supplementary Note 1) assuming no FRET but a bleed through (BT) contribution from doxorubicin fluorescence (red line and circles) and H1-EGFP FRET with the BT contribution from doxorubicin fluorescence (blue line and circles). The solid blue and red lines represent the simulated intensity-weighted mean lifetime values based on the median cell-wise measured contribution from doxorubicin fluorescence in the CEM (see Supplementary Note 1). The dashed red and blue lines indicate simulated values based on the 25th and 75th percentiles of the measured contribution from doxorubicin fluorescence. The magenta and green lines (measured intensity-weighted mean lifetime values) show the median (solid lines and circles) and 25th and 75th (dashed lines) percentile values of the distribution across all H1-GFP expressing cells and GFP expressing cells, respectively