Three-Dimensional Superresolution Imaging in Living Cells Using Single-Molecule A

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DESCRIPTION (provided by applicant): 3-D Superresolution Imaging in Living Cells with Single-Molecule Active Control Recent advances in microscopic imaging techniques with single molecules have led to superresolution information, that is, the ability to observe objects with resolution beyond the standard diffraction limit. These methods involve wide-field imaging, and require active control of the molecules in order to either turn emitters on or turn emitters off in order to maintain the concentration of emitters low enough to digitize the point-spread functions of individual molecules. By many imaging, photobleaching, and reactivation cycles, a superresolution image is obtained, but only for a two-dimensional projection of the actual three-dimensional sample. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), and have previously been applied primarily to fixed cells. However, many samples of biomedical interest, such as cells, are thick enough that two-dimensional imaging is a severe limitation. The primary goal of this research program is to achieve three-dimensional superresolution imaging in living cells using SMACM. This research will attack the problem of 3-D superresolution imaging with three thrusts. First, the optical illumination used to achieve active control will be tailored in its intensity as a function of time, in order to increase the efficiency of the reactivation and imaging process and eventually enable observation of time- dependent changes. Second, the microscope will be redesigned to utilize rotating point-spread functions. This relies on forcing the image of a single emitter to have a shape at the detector which rotates for different z- positions of the single molecule in the sample. The effect of this will be to enable much more precise determinations of the z positions of various single-molecule labels in the sample, which, when combined with precise localization in the x-y plane, will yield three-dimensional image information beyond the diffraction limit. Third, the research will implement multi-plane imaging in addition to rotating point-spread-functions, which will enable acquisition of 3D information over a greater depth into the sample. The results of this research will be to enable a new type of optical microscopy of cells, where three- dimensional superresolution information can be obtained in a noninvasive fashion about cellular substructures, including single molecules. The power of a single fluorophore as a nanoscale light source will then be used to its maximum benefit. By providing a new method for three-dimensional high resolution optical imaging in living cells, this research will bear directly upon biotechnological and biomedical applications as these fields currently utilize optical fluorescence microscopy of cells in many diagnostic situations. Current trends are pushing toward smaller and smaller spatial scales for analysis of the behavior and morphology of individual cellular structures. The ability to specifically and noninvasively analyze mutant or toxic behaviors of organelles and other tiny cellular structures will allow precise assessment of the utility of targeted drug treatments, which will help drive the future of medical interventions exactly at the point of disease.