Membrane binding by the CD3ε cytoplasmic domain in primary human T cells. B-A8 T cells expressing CD3ε-YF-TFP were placed into a flow cell and prepared for FLIM acquisition. All data are representative of n > 3 experiments. (A) Membrane binding by CD3ε_CD by FLIM in nonstimulated T cells. FLIM images of TFP (FRET donor) were taken before (basal FLIM) and after injection of R18 (FRET acceptor, 1 µg/ml, 90 s; FRET-FLIM). TCSPC counting was performed and processed using SPCImage. Fluorescence decay was calculated, and color-coded images were generated (color coding range, 1,000–3,000 ps). Shown are basal FLIM of TFP (left), FRET-FLIM after PM was labeled with R18 (middle), and R18 labeling of cells (right). Internal vesicles showed no differences in FLIM during R18 incorporation into the PM. Bars, 10 µm. (B) Relative distribution of mean fluorescence TFP lifetime per pixel at the PM ± R18 (basal FLIM, black line; FRET-FLIM, red line). (C) TFP fluorescence decay curves at PM in presence (red) or absence of R18 (blue). Single pixel TCSPC measurements were fitted to fluorescence decay curves using single exponential decay in SPCImage, minimizing χ² values. Increases in slope and χ² values indicate presence of multiple fluorophore states due to FRET. (D) TFP population analysis shows 20% decrease of overall fluorescence lifetime in presence of FRET acceptor; error bars represent SEM. n > 10 cells per experiment. (E) Examples of basal-FLIM and FRET-FLIM color-coded images taken from SPCImage software during analysis. Pictures show color-coded gradient of TFP fluorescent lifetime. Red ROI was used to generate graphs of relative distribution of fluorescence lifetime of photons. White crosshair indicates pixels that were used to generate fluorescence decay curves. Bars, 10 µm. (F) Fluorescence decay curves were calculated from single pixels, highlighted by white cross-hair in E, using either single or dual exponential decay algorithms, optimizing for low χ² values. Dual exponential decay calculations were made using a fixed long-lived component, set at 2,550 ps, based on mean TFP fluorescence lifetime in absence of R18 for a population of 30 cells (D). Mean fluorescence lifetimes (Tm), as well as short- and long-lived fluorescent lifetime components (T1 and T2, respectively), are presented. Also, relative abundance of short- and long-lived components (a1 and a2, respectively) is shown. Best fit for data are represented, and lowest χ² values are highlighted in red. For basal FLIM measurements, a single exponential decay function yielded an optimal χ² value (1.02), whereas a dual exponential decay function was optimal for FRET-FLIM (χ² of 1.06).