Fluorescence Recovery After Photobleaching (FRAP) is a method of determining the kinetics of diffusion in living cells (usually) using fluorescence microscopy. The general method is to label a specific cell component with a fluorescent molecule, image that cell, photobleach a small portion of the cell, then image the recovery of fluorescence over time.

Why is there recovery of fluorescence? Diffusion or active movement of molecules within the cell replace bleached fluorophore with unbleached molecules that were located in a different part of the cell. Over time fluorescence in the bleached region recovers.

Above are example curves of FRAP of the fluorescent dye Oregon Green. In this experiment the researchers conjugated Oregon Green to the cell surface receptor "Toll-Like Receptor-4". They watched the recovery of fluorescence after different treatments. Fluorescence recovery indicated that the TLR-4 molecule was movable within the plasma membrane.

Requirements for FRAP:

• "Moving" objects must be labeled with a fluorophore.
• Equipment must be able to bleach a defined area.

What does FRAP tell us:

• Diffusion of molecules
• Active movement of cell components
• Recycling of cell components
• FRAP is useful to look at how mobile a fluorescent molecule is. Is it free to move around or is it bound to something immobile? Is it bound to something big and slowly diffusing? Is it trapped in a particular space or compartment or excluded from a particular compartment?