FRAP

Fluorescence Recovery After Photobleaching (FRAP) is a quantitative microscopy technique used to measure the mobility of fluorescently labeled molecules in living cells, tissues, or model membranes. In a FRAP experiment, a defined region of interest containing fluorescent molecules is bleached rapidly by an intense laser, eliminating fluorescence in that region. The subsequent return of fluorescence is monitored over time as unbleached molecules move into the bleached area. The recovery curve reflects the combination of diffusion and binding/unbinding processes and can be analyzed to estimate parameters such as the diffusion coefficient, the mobile fraction, and, in some models, binding kinetics.

Procedure overview: samples are labeled with fluorescent tags (for example GFP fusions). A high-intensity laser is used to bleach a small region with minimal damage. Time-lapse imaging tracks fluorescence recovery. Data are normalized to pre-bleach intensity and corrected for overall bleaching during imaging. Common analyses fit the recovery curve to diffusion models (e.g., two-dimensional diffusion) to extract D and the mobile fraction. The t1/2 (time to half-maximal recovery) is often reported as a simple descriptor.

Applications include studying membrane protein mobility, cytosolic and nuclear protein dynamics, receptor trafficking, and protein–protein interactions in living cells. Limitations include photodamage, bleaching outside the ROI, and model-dependent interpretation; results reflect a combination of diffusion and transient binding, and may not distinguish these components without additional modeling or experiments. Variants such as FRAP with complementary methods (e.g., FLIP, FRAP-FLIM) extend the range of detectable dynamics.