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Fluorescence Recovery After Photobleaching (FRAP)
Fluorescence Recovery After Photobleaching (FRAP)
Photobleaching is the disappearance of fluorescent signal following the irreversible breakdown of fluorescent molecules after their interaction with molecular oxygen. In most live cell experiments, imaging conditions are optimized to minimize photobleaching, but deliberate photobleaching can prove a very useful technique. The observation and measurement of fluorescence recovery after photobleaching (FRAP) allows scientists to investigate the diffusion and motion of biological macromolecules. This is particularly useful for studying the mobility of fluorescently labelled proteins.
With FRAP, the fluorescently tagged protein of interest is visualized at low light intensity followed by photobleaching of a user-defined region of interest with high intensity light, causing the fluophores to bleach in the selected region.
3D Targeted Illumination
3D FRAP allows recovery to be visualized and measured in 3D offering new experimental possibilities. Bleaching is performed as part of an acquisition protocol so that pre- and post-bleach volumes are acquired. Targeting a specific plane in a 3D volume for high intensity illumination allows the unique possibility to observe post-bleaching of a 3D stack. This approach allows the creation of pioneering new experiments to provide greater understanding of life science research.
FRAP Experiment
The figure above shows a typical profile of fluorescence intensity collected during a FRAP experiment. Baseline fluorescence intensity is collected (A) before the photo-bleaching occurs (arrow) so that the amount of fluorescence is substantially reduced (B). After bleaching, fluorescence in the area increases as unbleached molecules diffuse into this area (C). A steady state is reached (D) when the recovery is complete. In practice, complete recovery is rare, as bound material is unable to diffuse out of the bleached region, this material is known as the "immobile fraction". The lateral mobility affects the slope of the curve (C). A steeper curve indicates a faster recovery, reflecting higher molecular mobility or diffusion coefficient.