Fluorescence Recovery After Photobleach
Many live-cell imaging techniques are designed to minimize the destruction of the fluorophore label however, FRAP measurements rely upon this process. Photobleaching is an irreversible process that involves the irradiation of the fluorophore with light in the presence of molecular oxygen. This results in the destruction of the fluorophore and with it it’s ability to emit fluorescence. In FRAP experiments, the area of photodamage is restricted and as the techniques’ name suggests, the recovery of fluorescence back into it is monitored. Recovery of the fluorescence signal is a result of the exchange of bleached fluorophores with those that are unbleached from the surrounding area. The fraction of molecules that are able and unable to be participate in this exchange are termed the ‘mobile fraction’ and ‘immobile fraction’ respectively. In addition to this, observing the rate of fluorescent recovery can provide important understandings of the movement and interaction of intracellular molecules.
FRAP measurements are typically performed on point scanning confocal microscopes, although spinning disk and widefield systems equipped with a dedicated bleaching laser can also be used. In a typical FRAP experiment low-power laser excitation is used to image the sample pre and post bleaching, during the bleach period, the region of interest is repeatedly scanned at high laser power until the original signal reduces by approximately 50%. The recovery period is then monitored using the original pre-bleach laser power.
The Nikon A1R confocal microscope is particularly suited to FRAP experimentation; galvo-scanning mode is utilized to target and perform the bleach while resonance-scanning mode is used to monitor the pre and post bleach fluorescence.
