Achieving 3D FRAP using multiphoton polygon scanning microscopy

Fluorescence recovery after photobleaching (FRAP) has been developed to measure molecular diffusion in living cells. However, conventional FRAP using a single stationary beam guided by a pair of galvanometer mirrors is not tailored for raster scanning microscopy. Furthermore, it has been shown that a single point of 2D FRAP only acquires molecular diffusion within a given imaging plane and does not fully capture the full molecular dynamics. Here, we address these limitations with a custom-built 2-photon polygon scanning microscope that features volumetric scanning with a frame rate of 20 fps and 170 nm pixel size. Importantly, our system allows photomanipulation to selectively measure FRAP from the diffusion dynamics of fluorescent molecules in a 3D sample. To demonstrate these capabilities, we performed rapid axial scans of fluorescent beads in suspension, achieving a volumetric scan rate of less than a second. FRAP functionality was verified in vitro on sulforhodamine-labelled giant unilamellar vesicles and diffusion kinetics determined from the rate of fluorescence recovery. The resolution and speed introduced from polygon scanning microscopy coupled with photomanipulation capabilities sets a precedent for 2-photon 3D FRAP imaging.