Fig. 6.

The plate illustrates photobleaching and photoswitching by 488 and 633 nm excitation of BCI photoactivated product in epoxy resin. We first activated the Bdnf gene expression-mediated BCI precipitate (10 μm in epoxy resin) with a single 720 nm scan (40x/1.3 NA objective, 6 mW laser power output) leading to moderate fluorescence after 488 (A) or 633 (B) excitation. We then focused on the area indicated with a white square (C) and scanned 50 times with maximal 488 nm laser power. Subsequently, we took a single image using 488 and 633 nm excitation (C, D). While 488 nm exposure had resulted in noticeable bleaching, this had no effect on the 633 nm-excited fluorescence emission (D). We subsequently restored the 488 nm-excited fluorescence with a single 720 nm scan, followed by 25 scans of 633 nm, again with maximum laser power. The bleaching nearly eliminated the 633 nm-excited fluorescence (white square in F), but substantially increased the 488 nm-excited fluorescence (white square in E). We followed this with another scan at 720 nm, which brought the 633 nm-excited fluorescence back to normal while quenching the 488 nm-excited fluorescence emission at the same time (compare E with H). Another 720 nm scan further bleached the 488 nm emission close to the initial emission (compare A with I), whereas the emission elicited with 633 nm became much stronger (compare B with J). All imaging settings remained constant throughout the experiment. Scale bar: 20 μm.