This Phase-I SBIR application, submitted in response to NIMH SBIR PA- 99-007, seeks to create a novel microscopy platform by combining total internal reflection (TIR) and fluorescent polarization techniques developed by Dan Axelrod (Michigan) with proprietary liquid crystal tunable filter and polarization devices developed at CRI, along with innovative imaging algorithms, to yield a near-video rate system with features ideally suited to investigating questions in neurobiology. Advantages: TIR provides exquisite spatial resolution (approximately 100 nm) and unique polarization states. Fluorescence polarization can determine translation, rotation, or orientation of labeled membranes and other structures, and the physical state of small ligands. Spectral imaging confers flexibility in the use of multiple fluorescent signals and FRET experiments. No similar system is available that offers these three major features. Goals: (a) Create a ready-to-use TIR-fluorescence polarization microscope system. (b) Incorporate proprietary CRI, Inc. technology, comprising polarization optics, tunable filters and innovative acquisition and processing algorithms. (c) Develop preliminary near-video rate control and acquisition software for sue in feasibility testing. Feasibility: In collaboration with neurobiologist Ron Holz (Michigan): (a) visualize membrane-folding and fusion events related to secretion in chromaffin cells at near-video rate, and (b) image individual GFP-labeled exocytotic granules and cytoskeletal elements during exocytosis.
The primary market for the imaging system developed in this project would be to researchers in neurobiology, pharmacology, drug discovery, and cell biology. Potentially large applications also exist in spectral karyotyping for a multi-spectral TIR-based imager that can distinguish 10 or more probes simultaneously.
