MULTISPECTRAL FP-TIR MICROSCOPY FOR NEUROBIOLOGY

根据NIMH SBIR PA- 99-007提交的第一阶段SBIR申请，旨在通过将Dan Axelrod （Michigan）开发的全内反射（TIR）和荧光偏振技术与CRI开发的专有液晶可调谐滤波器和偏振设备以及创新的成像算法相结合，创建一种新型显微镜平台，以产生具有理想功能的近视频速率系统，适合研究神经生物学问题。优势：TIR提供精致的空间分辨率（约100 nm）和独特的偏振态。荧光偏振可以确定标记膜和其他结构的平移、旋转或取向，以及小配体的物理状态。光谱成像赋予灵活性，在使用多个荧光信号和FRET实验。目前还没有提供这三个主要功能的类似系统。目标：(a)创建一个随时可用的红外荧光偏振显微镜系统。(b)合并CRI公司的专有技术，包括偏振光学、可调滤波器和创新的采集和处理算法。(c)为可行性测试开发初步近视频速率控制和获取软件。可行性：与神经生物学家Ron Holz（密歇根）合作：(a)以近视频速率可视化与染色质细胞分泌相关的膜折叠和融合事件，(b)在胞外作用过程中成像单个gfp标记的胞外颗粒和细胞骨架元件。拟议的商业应用：该项目开发的成像系统的主要市场将是神经生物学、药理学、药物发现和细胞生物学的研究人员。潜在的大应用也存在于光谱核型的多光谱红外成像，可以同时区分10个或更多的探针。

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. PROPOSED COMMERCIAL APPLICATION: 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.