FRAP consists of irreversibly bleaching fluorophores in a particular region of interest (ROI). To do this, the ROI was imaged at 100% intensity for all lasers (405, 458, 514, 561 and 633 nm). The fluorescence recovery is then monitoring with a 2% intensity laser (488 nm for DiO excitation and nm for PBd22-PEO14 -TMR excitation) and a confocal pinhole aperture of 3.1 µm. The rate of fluorescence recovery that represents the time needed for the fluorophores in the membrane that surround the ROI to diffuse into that region, re-equilibrating the local fluorescence intensity. FRAP experiments were performed on upper pole of GUVs (away from the coverslip), where a circular region of interest with a 5 ± 0.5 µm diameter was exposed to five bleaching scans at 100% laser power; the recovery was then monitored by recording a time series at a frame rate of 13 frames per second of size 256 × 256 pixels. The recovery curves were fitted with Origin Pro using the fluorescence recovery model of Soumpasis :