This proposal seeks to partially fund the purchase of a Leica SP8 3X pulsed stimulated emission depletion (STED) super-resolution microscope for the Light Microscopy Shared Resource at the University of Rochester Medical Center. Fluorescence light microscopy techniques coupled with an ever-expanding diversity of probes, provides an extremely powerful collection of tools for the interrogation of cell and tissue structure and function. Nevertheless, widely used techniques such as confocal and multi-photon microscopy are limited in their spatial resolution by the diffraction limit of light; practically this means that the size of objects smaller than ~200 nm are visualized as no smaller than 200 nm and multiple objects closer than 200 nm can not be resolved as individual distinct entities. In the past decade a variety of ?super-resolution? techniques have been commercially developed, which effectively break this diffraction limited barrier and thus permit visualization of fluorescence probes with unprecedented spatial resolution. As demonstrated within the proposal, the Leica STED microscope allows the collection of images from two fluorophores concurrently, yielding a 5-fold increase in lateral resolution and at least a 3-fold improved axial resolution. This enhanced resolution permits the visualization of molecules on distinct sub-cellular structures previously only resolvable by electron microscopy. The requested microscope is amenable to visualizing genetically encoded probes in live tissue and a wide variety of fluorescent probes using standard immunocytochemical techniques in fixed tissue. Our initial user group is diverse and consists of 10 major users and 14 minor users. This group represents users from many medical center academic units as well as the undergraduate campus and neighboring research institutions, representing a wide diversity of research interests including exocrine secretion, muscle disease, local signaling domains, inflammation, tissue engineering and cellular senescence. While these initial core users have varied scientific questions a common theme is that their scientific questions can not be adequately answered with instruments currently available on campus. This Leica STED microscope appears ideally suited to function as a state-of-the-art resource to assure these, and future investigators can leverage this technology to further the goals of their NIH funded research.
The proposal seeks funds to purchase an instrument which would allow users to capture images of structures inside cells that are too small, or too close together to visualize by conventional light microscopes. The microscope will be part of Shared Light Microscopy Resource of the University of Rochester Medical School and surrounding institutions and will be available to a wide variety of users to answer questions about cell structure and function relevant to normal heath and disease.
