DECONVOLUTION MICROSCOPE FOR LIVE CELL IMAGING

此申请要求资金购买最先进的应用精密三角洲视觉宽视场反褶积显微镜系统。活细胞成像正在成为索尔克研究所研究人员生物学研究的一个重要方面。该系统专门设计用于提供高分辨率的活细胞和组织分析，而没有共聚焦显微镜中使用的激光照明的负面影响。拟开展的研究涉及肿瘤抑制基因、遗传稳定性、蛋白质运输、与癌症相关的核内结构、病毒入侵、细胞运动、细胞分裂以及由谷氨酸、阿尔茨海默氏淀粉样蛋白和聚谷氨酸束介导的亨廷顿病中的神经元细胞杀伤。大多数研究需要时移成像、三维重建、使用两个或多个荧光团分析多个细胞以实现共定位、严格的温度控制和低强度光以避免光损伤和激活细胞应激反应。索尔克研究所目前缺乏一种具有所有这些功能的仪器。所要求的德尔塔电视系统的设计符合主要用户组的所有实验标准。反褶积显微镜采用标准的荧光显微镜，具有高度优化的宽视场照明。因此，不使用高强度激光照明，以避免可能损害活细胞或激活应激反应的光损伤，或可能影响定量分析的光漂白。此外，活细胞可以在延时实验中移动，导致感兴趣的区域离开被照亮的平面，但反褶积系统的宽视场和三维成像能力最大限度地减少了这个问题。最后，约束迭代反褶积是定量的，能最大限度地保留数据量，以最小的照度提供高灵敏度和分辨率。由于激发和发射滤光轮用于从标准hg蒸气灯中选择适当的波长，因此只需添加适当的滤光片即可添加新波长。这与共聚焦显微镜的有限激发能力形成对比，共聚焦显微镜的限制性激光线限制了可以可视化的荧光团，并且在紫外线下激发需要极其昂贵的紫外线激光器（这也是细胞毒性的）。所要求的仪器的灵活性、高动态范围和灵敏度、多细胞采样和快速三维成像特性将加速完成所提议的工作，这对癌症、病毒发病机制和神经系统疾病具有重要意义。

This application requests funds to purchase a state of the art Applied Precision Delta Vision wide-field deconvolution microscope system. Live cell imaging is becoming an ever important aspect of biological studies for Salk Institute investigators. The requested system is specifically designed to provide high resolution analysis of living cells and tissues without the negative aspects of the laser illumination used in confocal microscopy. The proposed research involves tumor suppressor genes, genetic stability, protein trafficking, intranuclear structures implicated in cancer, viral invasion, cell motility, cytokinesis, and neuronal cell killing mediated by glutamate, Alzheimer's amyloid protein, and polyglutamate tracts in Huntington's disease. The majority of the studies require time lapse imaging, three-dimensional reconstruction, analysis of multiple cells using two or more fluorophores to enable co-localization, strict temperature control, and low intensity light to avoid photodamage and activation of cellular stress responses. The Salk Institute currently lacks an instrument with all of these functions. The requested DeltaVision system has been designed to meet all of the experimental criteria of the major user group. Deconvolution microscopy employs a standard epifluorescent microscope with highly optimized wide field illumination. Thus, high intensity laser illumination is not used avoiding photodamage which can harm living cells or activate stress responses, or photobleaching which can compromise quantitative analysis. Further, living cells can move during a time-lapse experiment, causing the region of interest to leave the illuminated plane, but the wide field and 3-D imaging capacity of the deconvolution system minimizes this problem. Finally, constrained iterative deconvolution is quantitative, conserves the maximum amount of data, affords high sensitivity and resolution with minimum illumination. Since excitation and emission filter wheels are used to select the proper wavelengths from a standard Hg-vapor lamp, new wavelengths can be added by simply adding the appropriate filters. This is in contrast to the limited excitation capabilities of confocal microscopy where restrictive laser lines limit the fluorophores that can be visualized, and excitation in the UV requires an extremely expensive UV-laser (which is also cytotoxic). The flexibility, high dynamic range and sensitivity, multiple cell sampling, and rapid three-dimensional imaging properties of the requested instrument will expedite completion of the proposed work which has important implications for cancer, viral pathogenesis, and nervous system diseases.