Fig. 5. Use of photoactivatable-GFP to monitor Kv2.1 mobility. HEK cells were transfected with PA-GFP-Kv2.1HA and mRFP to mark transfected cells (mRFP not shown). Before activation, PA-GFP-Kv2.1HA-expressing cells displayed no GFP fluorescence (Preactivation). Photoactivation was via illumination of an ROI (red box) using a 405 nm laser for 500 milliseconds at 10-15% attenuation. This protocol resulted in bright green fluorescence with minimal photobleach. After activation, GFP was excited by 488 nm laser light (emission bandpass set at 505-530 nm) and monitored by scanning every 5 seconds for 250 scans (41 minutes). At 640 seconds post-photoactivation, green fluorescence is apparent on the cell membrane opposite to the activation ROI (red arrow). Note that even 2500 seconds following activation, most of the fluorescence is still restricted to the photoactivation ROI, consistent with the low recovery observed with FRAP. Bar, 10 µm.