Fig. 2. Two Photon FRET-FLIM detects HIV-1 Env conformations.
a Schematic representation of the FRET-FLIM system setup. Intramolecular FRET between Env trimer labels is resolved in sparse Env molecules whereas inter-molecular FRET is observed upon Env-Env association. Samples of HIV-1 virions in the presence or absence of T cells were imaged using a Leica DIVE two-photon system equipped with a pulsed two-photon laser tunned at 950 nm, a 100× magnification objective and photon-counting technology. The simultaneous intensity and lifetime measurements were obtained in 100 frames acquisitions (see material and methods for further details). b Micrographs showing images corresponding to intensity-based measurements obtained in two independent channels: donor emission (500-550 nm) and acceptor emission (600–650 nm) or to lifetime measurements of the donor. Scale bar 10 µm. Data from donor lifetime (ps) and calculated apparent FRET efficiency from intensity-based measurements is represented in kernel multi-parameter plots. In presence of both, donor and acceptor, FRET values experience a shift towards positive values concomitant with a drop in lifetime. (c–f) Two-dimensional (2D) kernel probability graphs showing FRET (FRET efficiency, Eapp) vs FLIM (Lifetime, in ps) data. High-density regions are depicted in darker grey color. c, d Plots corresponding to HIV-1 mature HXB2 V4-GFPOPT labelled with NbA488 and NbA594 nanobodies incubated without (c) or with (d) sCD4. e, f Plots corresponding to HIV-1 immature HXB2 V4-GFPOPT labelled with NbA488 and NbA594 nanobodies incubated without (e) or with (f) 10 µg/mL concentration of soluble CD4 (sCD4D1-D4).