(A) Schematic of the experiment. A bottom pole of a micromanipulated GUV is placed in the focal plane of the confocal microscope. The fluorescence in a disk-shaped region of interest is monitored before bleaching and during the recovery phase. The three pictures show the pole of the GUV delineated by the red circle, the region of interest delineated by the white circle (here, the diameter is 5 µm) before bleach, right after bleach and after recovery from left to right respectively. The graph shows the raw fluorescence recovery in the region of interest (blue points), the three data-point rolling average (orange) and the fit of the rolling average using the equation indicated in the Materials and methods section. (B) The square of the diameter of the region of interest is plotted vs. the raw values of τ (multiplied by a factor of 16), from the fit of 6 different FRAP experiments (three experiments performed twice on two different days). The slope is equal to the diffusion coefficient, D, here 0.69 µm²/s. At the bottom, the extreme values of D obtained from the data point (red lines) are indicated. Hence, D = 0.7 ± 0.3 µm²/s.