Clathrin- and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors. However, due to their dimensions are within the diffraction limit, traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane. In this study, we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation (T-Rex) stimulated emission depletion (STED) super-resolution microscopy. With this line-switching mode, the cross-talk between the two channels, the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated. The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance. With this super-resolution microscope, not only the ring-shaped structure of clathrin and caveolae endocytic vesicles, but also their semi-fused structures underneath the cell membrane were distinguished clearly. The resultant information will greatly facilitate the study of clathrin- and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex STED microscope with this line-switching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.
Resolving the spatial relationship between intracellular components by dual color super resolution optical fluctuations imaging (SOFI)