Enhanced scanning control in a confocal scanning laser microscope

AbstractA fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user‐selected closed curves. The scanning unit uses two galvanometer‐mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time‐resolved recordings within the millisecond range can be performed. Repetition rates up to > 1 kHz for line scanning and curve scanning, and > 100 kHz for point scanning are obtainable. This allows time‐resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal‐to‐noise ratio obtainable with a point scanning confocal microscope.