Fluorescence Recovery after Photobleaching (FRAP)
Cells were immobilized as described above. FRAP experiments were
performed using a Marianas Yokogawa-type spinning disc inverted confocal
microscope with a 100x/NA1.45 oil (Alpha Plan-Fluar) objective. The
system featured fast shutter speeds and channel switching for high-speed
imaging and a vector high-speed point scanner for bleaching, controlled
using Slidebook software. For each experiment, we first identified a
well-immobilized cell and verified the fluorescence signal with a
~100-500 ms exposure. We then used the time-lapse mode
to capture images with the fastest camera speed for a pre-photobleaching
record and then used the screen selection tool and real-time imaging to
draw a ROI (region of interest) for photobleaching.