DECONVOLUTION MICROSCOPE FOR LIVE CELL IMAGING

这个应用程序要求资金购买最先进的应用精密Delta Vision广域反卷积显微镜系统。对于索尔克研究所的研究人员来说，活细胞成像正在成为生物学研究的一个永远重要的方面。所要求的系统是专门设计的，以提供对活细胞和组织的高分辨率分析，而不会出现共焦显微镜中使用的激光照明的负面影响。拟议的研究涉及肿瘤抑制基因、遗传稳定性、蛋白质运输、与癌症有关的核内结构、病毒侵袭、细胞运动、胞质分裂以及由谷氨酸、阿尔茨海默氏症淀粉样蛋白和多谷氨酸束介导的神经元细胞在亨廷顿病中的杀伤。大多数研究需要时间推移成像、三维重建、使用两个或多个荧光团对多个细胞进行分析以实现共定位、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DeltaVision系统的设计符合主要用户群体的所有实验标准。去卷积显微镜采用标准的荧光显微镜，具有高度优化的广域照明。因此，不使用高强度激光照射，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，导致感兴趣区域离开照明平面，但去卷积系统的宽视场和3D成像能力将这个问题降到最低。最后，约束迭代反褶积是定量的，保留了最大的数据量，在最小照度下提供了高的灵敏度和分辨率。由于激发和发射滤光器轮用于从标准汞蒸汽灯中选择合适的波长，因此只需添加适当的滤光器即可添加新的波长。这与共焦显微镜有限的激发能力形成对比，在共焦显微镜中，受限的激光线限制了可以可视化的荧光团，而在紫外光中激发需要极其昂贵的紫外光激光(这也是细胞毒性的)。所需仪器的灵活性、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拟议的工作，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.