Red Española de Microscopía Óptica Avanzada
Photobleaching Techniques/ FRAP-Fluorescence Recovery After Photobleaching
Consejo Superior de Investigaciones Cientificas (CSIC), Instituto de Biomedicina y Biotecnologia de Cantabria - IBBTEC - Servicio de Microscopia Optica Avanzada IBBTEC
Photobleaching techniques are used as a strategy to reveal the diffusion/binding dynamics of molecules or the exchange between compartments in live cells, tissues or organisms. The most straightforward photobleaching technique is the Fluorescence Recovery After Photobleaching (FRAP). In FRAP, fluorescent molecules are deactivated in a region of interest by focused light (tipically a laser beam) and the recovery of the fluorescence intensity in that region is followed in time, quantified by suitable numerical analysis (normalization and curve fitting), and compared to the initial conditions.

Fluorescence recovery into the region is due to the diffusion of the molecules in and out the region. Bleached molecules, which can diffuse out of the region, are substituted by other unbleached molecules that diffuse inside the region from surrounding areas. Quantification and analysis of fluorescence recovery provide information of descriptive parameters such as mobile and immobile fraction and half time of recovery.
Further analysis by kinetic modelling provides information on the molecular dynamics such as the diffusional properties of the studied molecule and its binding characteristics.
Equipment for this technology