Part A displays a phasor plot where data points cluster on a universal semicircle with a phase lifetime (tau(phi)) of 2.57 ns before W S C treatment, including an inset of a circular fluorescent cell membrane. Part B shows the same phasor plot and inset five minutes after the application of water-soluble cholesterol (W S C), with the phase lifetime remaining constant at 2.57 n s. Part C presents a time-course graph tracking the lifetime of G R A M-W-e G F P in nanoseconds over 15 minutes, with green square data points remaining stable near 2.6 n s. Statistical significance is noted as n.s. (not significant) for all time points, confirming that cholesterol enrichment does not statistically alter the fluorescence lifetime of the G R A M-W-e G F P probe. All values are approximated.
WSC treatment does not change the fluorescence lifetime of eGFP-GRAM-W. (A and B) Phasor analysis of eGFP-GRAM-W at the PM of tsA cells before and 5 min after WSC treatment. Plots depict fluorescence distribution and the corresponding apparent phase lifetime (τφ). (C) Time course of the mean phase lifetime (τφ) shows no change over 15 min of WSC treatment (mean ± SEM, n = 5 cells; n.s., not significant using a paired t test). During this period, eGFP-GRAM-W exhibited a decrease in fluorescence anisotropy and an increase in intensity, consistent with sensor clustering and increased local density. In contrast, restriction of GFP mobility due to aggregation would be expected to increase anisotropy. Moreover, the stability of the fluorescence lifetime under these conditions rules out a change in rotational mobility as the cause of the anisotropy decrease. Instead, these data are indicative of fluorescence depolarization via an increase in homo-FRET efficiency.