Strong laser power induces GP decrease and masks the effect of insulin in the local membrane environment of the insulin receptor but not ACP-GPI. Time course of mean delta GP change compared to time 0. (A–C) After 2 h serum starvation, cells stably or transiently expressing 2031-ACP-IR were labeled with CoA-PEG5-NR (A and B), or cells transiently expressing ACP-GPI were labeled with CoA-PEG11-NR (C). Spectral imaging was performed using different laser powers. Control solution or insulin solution were treated at 120 s (A; weaker laser power) or at 180 s (B; stronger laser power). Control solution and insulin solution were added at 60 and 240 s, respectively (C). Data represent the mean ± SEM from 11 time-lapse image sets for control, 13 for insulin of weaker laser powers (A), 10 for both control and insulin of lower laser power (B), and 9 for 1.5% of lower laser power and 8 for 4% laser power (C).