Super resolution fluorescence microscopes greatly expand our ability to study from cellular level down to single molecule level. All existing super-resolution methods have their pros and cons. There lacks a technique that combines single molecule sensitivity, large field of view, fast imaging speed and minimal photon toxicity. This research proposes to develop a nonlinear structured illumination microscopy (SIM) method that utilizes stimulated emission depletion (STED) instead of saturation to break the diffraction limit. STED-SIM will achieve single molecule sensitivity as in single fluorophore super resolution methods, such as PALM/STORM. It will double the field of view of point-scanning STED at the same image speed, retain a high dynamic range as SIM, and reach a resolution of 20~30 nm, a factor of 2 better than saturated structured illumination microscopy (SSIM). STED-SIM will operate at the total internal reflection (TIRF) mode, in which the evanescent excitation and STED field will not cause unnecessary photon toxicity. With its fast speed, high sensitivity, full field of view, and minimal photo toxicity, STED-SIM may provide a better solution for live super-resolution imaging of membrane resident or near membrane structure, and allow precisely following questions related to cell-matrix and cell-to-cell coupling, as well as near membrane cell signaling. In this research we will demonstrated STED- SIM's ability through two imaging experiments: single DNA molecules, and the organization of cell matrix interactions via integrin mediated focal contacts.
The research will develop a fast, sensitive, full field super resolution microscope for live imaging cell-matrix and cell-to-cell coupling, as well as near membrane cell signaling. The instrument will help improve our understanding in cancer growth and development etc.
