Fluorescence recovery after photobleaching (FRAP) is a technique that uses a confocal microscope to study the diffusion of molecules within a membrane. During FRAP, fluorescently-labeled molecules within a specified region of interest (ROI) are photobleached using a high-power laser. After photobleaching, the fluorescent intensity in the ROI is reduced to zero. Due to molecular diffusion that naturally occurs within the membrane, surrounding fluorescent molecules will move into the bleached area. The confocal microscope is used to visualize the recovery of fluorescent intensity within the ROI over time. Analysis of the recovery curve must be performed to extract meaningful quantitative data, such as the diffusion coefficient and the halftime recovery of the sample. Having insight on these properties is crucial for understanding molecular diffusion within a given material. Commercial confocal microscopy software can perform FRAP, yet it lacks the analytical tools to extract these properties. Therefore, in this project, a user-friendly interface was developed in MATLAB to curve fit the data, extract the diffusion coefficient and halftime recovery, and visualize the confocal images. Proof of concept testing using FRAP data of insulin diffusion through a polymer capsule demonstrated the efficacy of the interface.
