Fluorescence recovery after photobleaching (FRAP) and analysis

FRAP experiments were conducted with the Spinning Disc Ultraview VOX confocal microscope using the ‘UltraVIEW PK Device’ as a bleaching device through a 488 nm laser channel. For in vitro droplets, ‘UltraVIEW PK Cycles’ was set to 20, ‘Spot Cycles’ was set to 20, and ‘Spot Period’ was set to 80 ms. Images before bleaching were captured for 10 time points at a rate of 1 timepoints per second. Recovery images were captured for 500 s at a rate of 5 s per timepoint. For cellular droplets, ‘UltraVIEW PK Cycles’ was set to 100, ‘Spot Cycles’ was set to 100, and ‘Spot Period’ was set to 5000 ms. Images before bleaching were captured for 5 time points at a rate of 1 timepoints per second. Recovery images were captured for 120 s at a rate of 1 s per timepoint. Analyses were performed using ImageJ and graphs were plotted using GraphPad using the one-phase association model.