Fig. 3. ixFLIM FRET demonstration on internally labelled Cy3–Cy5 DNA constructs.

a Schematic geometry of the dye attachment to the DNA. The numbers indicate the Cy3–Cy5 mutual distance in base pairs (bp). Chemical structure adapted from https://en.wikipedia.org/wiki/File:Cy3_Cy5_dyes.gif by "Nwbeeson". b Cy3 (green, shorter wavelength) and Cy5 (red, longer wavelength) absorption (light shade) and emission (dark shade) spectra, overlaid by the broadband excitation pulse spectrum (black), truncated by a 700 nm short-pass filter to facilitate excitation of both donor and acceptor and detection of longer-wavelength Cy5 emission only. c FLIM image $FLIM\left(x,y,⟨t⟩\right)$ of the beads to which the DNA constructs are attached with 20 bp Cy3–Cy5 distance. d ixFLIM transient map $ixFLIM\left(t,{\lambda }_{\tau }\right)$ obtained from the image in (c). e Transients in the donor and acceptor spectral region (as identified spectrally according to (b), overlaid with fits from global analysis of the ixFLIM data from (d). f Spectral components from the global analysis, 240 ps rise of the donor signal due to FRET (black), 1.34 ns decay of the emitting acceptor (red). g Spectral fit (orange) to extract E according to Eq. (6), decomposing the raw excitation spectrum (i.e., not divided by the laser spectrum, blue) into the weighted sum of the donor and acceptor absorption spectra multiplied by the laser spectrum. Fit residuals are shown in gray. h A complementary standard full-frame FLIM measurement with 530 nm excitation and detection of donor emission. Shown is the donor emission decay in the presence of acceptor (red) and with acceptor bleached (black). The curves are overlaid with fits (dashed) as described in the SI, fit residuals are shown as well (dotted). The number of experimental repetitions is described in the Statistics and Reproducibility section in Methods.