FRET acceptor-photobleaching experiments

Microscopy: Experiments were performed on an Olympus IX81 inverted fluorescence microscope equipped with a 60× UPlanFLN 0.9 NA objective, a photomultiplier tube (Hamamatsu Photon Counting Head H7421-40, Hamamatsu City, Japan) and a 100 mW 515 nm laser (Cobolt, Sweden) that was coupled into the system via an AHF F73-014 z514 DCRB notch filter (Supplementary Fig. ^2a). Data acquisition from the PMTs was performed as described by Sourjik et al.³⁵. Fluorescence was excited with a MT20 illumination system. In order to attenuate CFP and minimize bleaching, the internal neutral density (ND) filter of the MT20 was set to 7.72%, and further reduced by an external (ND = 2) filter. A dense multilayer of cells was illuminated with excitation light centered around the CFP excitation maximum (EX: 438/24 nm, Dual BS 440/520 nm, EM: 475/23 nm) for the entire experiment to achieve continuous bleaching of CFP and avoid recovery effects. Prior to acceptor photobleaching, CFP emission signals were measured for 60 s. The sample was then defocused by −16 µm to increase the bleaching area of the laser. The acceptor was bleached with the laser at maximum power for 20 s. After refocusing the sample, CFP emission was recorded for another 60 s. The efficiency of acceptor photobleaching was monitored by recording YFP fluorescence (EX: 504/12 nm, Dual BS 440/520 nm EM: 542/27 nm) for 6 s before and after each experiment. Furthermore, after bleaching, a YFP image (EX: 504/12 nm, Dual BS 440/520 nm EM: 542/27 nm, 100% illumination intensity, 3 s exposure) was taken with a EMCCD Hamamatsu C9100-2 camera to check for homogenous bleaching of the sample area.