Figure 3.

TZM Forster Resonance Energy Transfer (FLI-FRET) microscopy (FLIM-FRET) analysis in AU565 cancer cells. (A) The representative time-correlated single photon counting (TCSPC) images of fluorescence intensity and mean lifetime map (τ_m) in cells treated with TZM-AF700 only [Acceptor/Donor (A:D) = 0:1; donor single-labeled] or with TZM–AF700 plus TZM–AF750 (A:D = 2:1; double-labeled); pseudo-color range = 500–1500 ps. Zoomed regions of interest (ROIs) of both single and double-labeled cells show heterogeneity of fluorescence lifetime of TZM–AF700 within the cells. White arrows indicate x, y coordinates used for the curve fitting using SPCImage. Scale bar = 50 µm. (B) Representative fitting curves and IRF, the fluorescent lifetime decay in the single and double-labeled cells was determined by comparing the fitting of the decay data using both single- and double-exponential decay models, respectively. (C) Fluorescent lifetime distribution in AU565 cells treated with TZM–AF700 (A:D = 0:1) or TZM–AF700 and TZM–AF750 (A:D = 2:1). (D) Quantification of FRET donor (FD%) levels in AU565 cells treated with a near-infrared (NIR) TZM–FRET pair at various A:D ratios. Analysis was performed using 10 distinct pixel coordinates (n = 10) from five independent ROIs; error bars represent confidence interval at 95%. (E) Quantification of TZM–FRET efficiency (E) in relation to A:D ratios. Data presented as mean ± confidence interval at 95%, n = 10.