Rectangle FRAP for Measuring Diffusion with a Laser Scanning Microscope

Fluorescence recovery after photobleaching (FRAP) is one of the most useful microscopy techniques for studying the mobility of molecules in terms of a diffusion coefficient. Here, we describe a FRAP method that allows such measurements, relying on the photobleaching of a rectangular region of any size and aspect ratio. We start with a brief overview of the rectangle FRAP theory, and next we provide guidelines for performing FRAP measurements, including a discussion of the experimental setup and the data analysis. Finally, we discuss how to verify correct use of the rectangle FRAP method using test solutions.