Fluorescence Recovery After Photobleaching (FRAP)

Abstract Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscope technique to measure molecular diffusion and transport. FRAP is a valuable technique in cell biological research and evolved conjointly with microscope and fluorescent probes advancements. Although developed in the 1970s, the discovery and further development of fluorescent proteins revolutionised FRAP. After the discovery of green fluorescence protein and its application as a noninvasive and genetically coded protein‐tag, in vivo studies of protein dynamics and interactions became possible. FRAP is based on irreversibly bleaching a pool of fluorescent probes and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. Although measurements are straightforward, quantitative FRAP requires careful experimental design, solid controls, data collection, and analysis. Over the past years, several FRAP‐related techniques have been tailored to suit particular cell biological questions, including inverse FRAP, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching. Key Concepts: Fluorescence recovery after photobleaching (FRAP) is a method to qualitatively and quantitatively study biomolecule dynamics in living cells. FRAP is based on irreversibly bleaching a pool of fluorescent probes with high intensity light and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. FRAP experiments are often conducted on confocal microscopes. To derive quantitative results from such experiments, several parameters and controls need to be considered and utilised in the analysis. There are several FRAP‐related methods that have been developed for specific applications and biological questions. FRAP is a versatile and popular method in modern biomedical research. Its application is broad and is increasingly applied in pharmacological, therapeutic and diagnostic areas.