Figure 4.

FRET trajectory analysis of HIV-1 particles containing donor-labeled IN and acceptor-labeled IN using particle-based pixel binning. (A) Phasor analysis of viral HIV-1 particles containing both donor- and acceptor-labeled IN (IN-Turquoise2+IN-mVenus) forming an unknown mix of multimers including monomers and dimers and potentially higher-order multimers. The donor-only (DO) phasor (g = 0.82, s= 0.37 and background (BG) phasor (g = 0.78, s= 0.31) determine the starting point and end of the FRET trajectory. Variations of the donor-only signal contribution in the analyzed particles result in a variety of FRET trajectories and FRET trajectory end points (red dotted line). (B) Phasor analysis of viral HIV-1 particles containing both donor- and acceptor-labeled IN (IN-eGFP+IN-mScarlet) forming an unknown mix of multimers including monomers and dimers and potentially higher-order multimers. The DO (g = 0.90, s = 0.29) and BG (g = 0.79, s = 0.31) determine the starting point and end of the FRET trajectory. Variations of the donor-only signal contribution in the analyzed particles result in a variety of FRET trajectories and FRET trajectory end points (red dotted line). In both (A) and (B) the pure monoexponential lifetime of the donor (${\tau }_D$) and BG determine by intensity fraction the position of DO on their connecting fraction line (green dotted line). (C) FRET histogram for (B) extracted from the 0, 10, and 20% passive donor contribution FRET trajectories of which the 0 and 20% histogram averages are indicated by purple dashed lines and the 10% passive donor histogram (shown) average is depicted as a vertical full red line with its value.