This project is the development of a fluorescence hyperspectral microscope. A hyperspectral microscope collects a broad region of the spectrum with high spectral resolution in every sampled spatial volume. This microscope will improve and optimize the design of a Sandia National Laboratories designed hyperspectral microscope that incorporates a high throughput prism spectrometer and state of the art electron multiplied CCD cameras. Optimizations will include a focus on speed, with a target of full spectral imaging at 30 Hz, and will be based on a commercial inverted microscope to allow standard accessories and practices used for live cell imaging. High spectral resolution will allow separation of up to eight spectrally distinct quantum dots, which due to their broad excitation spectra can be excited simultaneously with a 488 nm laser. Spectral separation combined with the ability of single particle tracking to localize single particles to less than 10 nm will allow the observation of either hetero or homo protein-protein interactions at length scales close to 10 nm, much less than the 250 nm resolution of a fluorescence microscope. The project will begin with a redesign of the Sandia hyperspectral microscope to incorporate line scanning for optimal speed. Analysis techniques will be developed to utilize knowledge about the single particle, diffraction limited spatial signature of the probes to enhance spectral separation and localization. The first application will be to study the dynamic homo-interaction between the membrane protein FceRI. The microscope will be developed and housed in a laboratory in the University of New Mexico physics department, and after construction, is expected to be heavily used by researchers of the UNM cancer centers. Public Health Relevance Statement: This project will fund the development of a 'fluorescence hyperspectral microscope'that will make use of new fluorescent probes (quantum dots) and spectral multiplexing to observe the dynamics of protein-protein interactions at the 10 nm scale. Data generated with this instrument is expected to give new insight into cellular signaling pathways, including those involved in the immune system and cancer.
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