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[How FRAP works] [Photobleaching] [Molecule Diffusion] [Recovery Dynamics] [Fluorophores]

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The method of fluorescence recovery after photobleaching (FRAP) utilizes the phenomenon of photobleaching of fluorescent probes to measure parameters related to molecule mobility. It was initially designed by Axelrod and coworkers to measure two-dimensional diffusion of membrane-bound molecules (see applications of photobleaching techniques for further application details ).

Frequently photobleaching interferes with image acquisition in fluorescence microscopy by fading the fluorescent probes, resulting in a lower signal/noise ratio. However in FRAP experiments, it is used to selectively photobleach a specific area by high intensity laser pulses. Subsequently the kinetics of fluorescence recovery are recorded by sampling images at regular time intervals with low intensity illumination.

Closely related to FRAP is fluorescence loss in photobleaching (FLIP). In FLIP experiments a specified region of the cell is repetitively photobleached and the loss of fluorescence in non-bleached parts of the cell is measured.

Another variant of photobleaching techniques is iFRAP (inverse FRAP) where all the fluorescent molecules in a cell except for a small region are bleached . The loss of fluorescence from the unbleached region in the postbleach images is then analyzed. With this technique qualitative information about mobility and equilibration time can be gained. Because of the time needed to bleach large areas, this method is especially suited to analyze the dissociation parameters of molecules which are bound to an immobile structure for several seconds.

Read more about the theory behind FRAP, needed equipment or how to perform a FRAP experiment.

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contact: Stefan Terjung           last update: 02/06/04