Fluorescence recovery after photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. The resulting information serves to determine kinetic properties such as the diffusion coefficient, mobile fraction, and transport rate of the fluorescently labeled molecules. FRAP employs irradiation of a fluorophore with a short laser pulse. State of the art laser scanning microscopes such as the Leica TCS SP8 have the advantage of using a high intensity laser for bleaching and a low intensity laser for image recording. The LAS AF application wizard offers different ways to carry out a FRAP experiment. Timing parameters can be adapted to various experiments, e.g. moderate, fast, or multi-step kinetics. Moreover, related experiments such as photoactivation and photoconversion or FLIP experiments are possible.
For very fast kinetics, FlyMode can be applied. Using FlyMode, the reading out of signals during the x fly back of the scanner provides a time resolution between lines – instead of between frames – for the FRAP experiment. Depending on the necessary bleaching power you may choose ROI bleach or ROI with Zoom In bleach combined with one or multiple bleach steps. A free y format reduces scanning time during bleaching when multiple bleach intervals are needed.
With the introduction of the Leica TCS SP8, the FRAP wizard offers improved functionality and additional features: