Fluorescence recovery after photobleaching (FRAP)

About this technique

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Fluorescence recovery after photobleaching (FRAP) involves bleaching a defined area of a sample with high-intensity light and then recording the recovery of fluorescence into the bleached area using a lower light intensity (low enough to record a signal, but not to induce further bleaching). The recovery of fluorescence into the bleached area then results from movement of unbleached fluorophores from surrounding areas moving into the bleached region. The rate of this recovery can be used to estimate the mobility of molecules and to determine, diffusion, active transport, synthesis or natural turnover of those molecules. An alternative technique measures the decrease in fluorescence in surrounding areas to determine these rates. This is called fluorescence loss in photobleaching or FLIP.

FRAP can be used to determine whether there is transport between different cellular compartments within a cell. For example, FRAP may be able to determine:

• if molecules are moving from or between organelles, or from the cytoplasm into the nucleus.
• if molecules are relocating within a cellular membrane.
• if molecules are being incorporated into other molecules.
• the speed at which molecules are moving.

As FRAP is measuring an active process, experiments are usually carried out on live samples. Special care must be taken the ensure samples are prepared in such a way that measurements most accurately represent activity in the normal cellular environment. 

Confocal microscopes are usually used for FRAP experiments as they allow very accurate definition of the region of interest which is to be bleached. Additionally, all modern confocal microscopes have very precise control over input powers and, therefore, bleaching and subsequent measurements can be accurately controlled. This is very important since excessive bleaching can cause molecular alteration and cellular damage resulting in meaningless data.