A DECONVOLUTION MICROSCOPE FOR CELL BIOLOGICAL RESEARCH

我们请求资助购买一个集成的数字反褶积显微镜工作站，以检查固定细胞和活细胞中蛋白质、染色体、细胞器、病毒和细胞骨架元件的表达和定位。有了新的数字反褶积显微镜，普林斯顿大学的研究人员将能够以前所未有的分辨率（优于0.2微米）和清晰度对生物标本进行光学切片。反褶积显微镜可以通过使用计算方法从其他焦平面上数字化地去除“失焦”光，从而达到这种出色的分辨率水平。光学切片可以将它们组合在一起，产生高精度的亚细胞结构三维模型。与快速和极其敏感的数码相机（能够检测到单个光子）相结合，反褶积显微镜将允许研究人员使用快速延时成像来跟踪活细胞内的快速动态过程。该建议详细介绍了数字反褶积将产生重大影响的四个主要项目和一个次要项目。这些项目包括：(1)阐明在位点特异性有丝分裂重组过程中发生的长距离染色体相互作用，例如在酵母中发生的交配型相互转换；(2)可视化疱疹病毒在神经元细胞中的侵袭和传播；(3)以酵母为模型系统分析微管定向和动力学对核定向和迁移的作用。(4)可视化端粒和核包膜，研究核外周定位对端粒转录抑制的作用；(5)分析活酵母细胞的分泌途径，测试不同的高尔基生物发生模型。在每种情况下，由于细胞和/或其内部结构的极小尺寸，这些项目需要独特的光学切片能力和数字反褶积显微镜的分辨率。通过这些手段，数字反褶积显微镜将为生命科学中使用的最强大的光学显微镜之一提供杰出的研究和教育机会。

We request funding for the purchase of an integrated digital deconvolution microscope workstation to examine the expression and localization of proteins, chromosomes, organelles, viruses and cytoskeletal elements in fixed and living cells. With the new digital deconvolution microscope, researchers at Princeton University will be able to optically section biological specimens with unprecedented resolution (better than 0.2 micron) and clarity. The deconvolution microscope can achieve this outstanding level of resolution by using computational methods to digitally remove "out-of focus" light from other focal planes. The optical slices can them be combined to produce highly accurate three-dimensional models of sub-cellular structures. Coupled to fast and extremely sensitive digital cameras (able to detect single photons), the deconvolution microscope will allow researchers to follow rapid dynamic processes within living cells using fast time-lapse imaging. This proposal details four major, and one minor, projects where the digital deconvolution will have a major impact. These projects include: (1) elucidation of the long range chromosome interactions that occur during site-specific mitotic recombination such as occurs during mating type interconversion in yeast, (2) the visualization of Herpes virus invasion and spreading in neuronal cells, (3) analyzing the roles of microtubule orientation and dynamics on nuclear orientation and migration using yeast as a model system, (4) visualization of telomeres and the nuclear envelope to examine the role of nuclear peripheral localization on the transcriptional repression that occurs at telomeres, and (5) analysis of the secretory pathway in live yeast cells to test different models of Golgi biogenesis. In each case, because of the extremely small size of the cells and/or of their internal structures, these projects require the unique optical sectioning power and resolution of the digital deconvolution microscope. By these means, the digital deconvolution microscope will provide outstanding research and educational opportunities on one of the most powerful optical microscopes available for use in the life sciences.