The ongoing quest to delve ever deeper into a living cell has brought new insight into dynamic biological processes. But conditions inside a cell, including crowds of individual molecules, often flout the best efforts of modern imaging technology to observe activity. And that’s not to mention the fact that cells become unhealthy and die during time-lapse imaging.
So Vladislav Belyy and his biophysics colleagues at UC Berkeley sought to overcome some of these issues as they developed their new PhotoGate microscopy technique, which Belyy introduced Monday at the microsymposium on spatial organization of the cell.
Using a pair of lasers, the PhotoGate technique involves pulsing one laser as a gate beam to sweep outwards from the center of a region of interest, creating a photobleached circular area. That laser then is shuttered for several seconds to let a few unbleached molecules diffuse back into the area. The second laser, called the imaging beam, is used to image fluorescent molecules in the area. To prevent too many fluorescent molecules from diffusing back into the region, the gate beam sweeps repeatedly along the region’s outer perimeter.
“You can track particle trajectories on the order of tens of seconds,” Bedlyy said, a sharp increase from what previously could be done to make molecular movies “With PhotoGate, you can see the arrival and departure of single proteins in organelles.” Plus, he noted, PhotoGate works well for multicolor applications because it uses the same wavelength for both gating and imaging. And it can be used on existing cell lines; some previous technologies required developing new strains specifically suited to single-molecule imaging.