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.
Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.
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