Deltavision RT Image Restoration System for Established Research Core Facility

描述（由申请人提供）：俄勒冈州健康与科学大学的MMI研究核心设施是一个完善的共享资源设施，为OHSU和波特兰市区26个部门的80多个实验室提供高分辨率荧光显微镜。该申请要求提供资金，以便用新的API DeltaVision RT系统（DVRT）替换过时的Applied Precision DeltaVision图像恢复系统，该系统具有活细胞和激光增强应用所需的配件。DVRT将用于补充成像与激光扫描共聚焦显微镜（LSCM）提供给我们的核心设施。所要求的DVRT将用于对粘附细胞厚度小于~50 μ m且荧光相对较弱的细胞培养样品进行成像。对于这些标本，DVRT的迭代约束反卷积图像恢复超过LSCM成像的分辨率和信噪比。所要求的DVRT已经显示出其本身对于易受光损伤的细胞或待成像的荧光分子易受光漂白或暗淡的情况的上级，在实验中，两种或更多种颜色的荧光标记分子相互作用，用于获取微分干涉对比度（DIC）沿着荧光，以及用于多个数据集和3D和4D重建的高度精确的点访问。所要求的DVRT提供的新功能包括比我们当前系统快3-4倍的颜色切换;比我们目前使用客户提供的低水平荧光样品所能实现的信噪比（S/N）高10- 20倍;受控环境室;改进的极精确XYZ载物台，具有自动聚焦功能，可在每个z堆栈（沿Z轴沿着的一组光学部分）内保持聚焦。这对于延时研究很重要，因为在延时研究中，载物台漂移和其他机械或生物来源会导致长时间（数小时至数天）的焦点丢失。所要求的辅助二极管激光器提供进行荧光共振能量转移（FRET）、（激光诱导）光漂白后荧光恢复（FRAP）和光漂白中荧光损失（FLIP）所需的聚焦照明，以研究活细胞中特定蛋白质和亚细胞组分的动态相互作用。特别是，DVRT系统已被证明远远上级任何其他可用于超大文件（>2GB），需要3D或4D重建，以正确理解数据。相关性-我们的成像设施的26个部门的80多个客户进行基础生物医学和转化医学研究。例如，研究致病微生物逃避人类宿主检测的生存机制，神经系统中的细胞间通讯和控制，淋巴细胞和抗原呈递细胞之间免疫突触的形成，人类病毒的细胞间传播，以及鼠伤寒沙门氏菌、土拉弗朗西斯氏菌、淋病奈瑟氏菌和奈瑟氏菌的发病机制和宿主细胞存活。脑膜炎

DESCRIPTION (provided by applicant): The MMI Research Core Facility at the Oregon Health & Science University is a well-established shared resource facility providing high-resolution fluorescence microscopy to more than 80 labs in 26 departments at OHSU and Portland metro area. This application requests funds to replace an outdated Applied Precision DeltaVision image restoration system with a new API DeltaVision RT system (DVRT) with the accessories needed for live-cell and laser enhanced applications. The DVRT will be used to complement imaging with laser scanning confocal microscopes (LSCM) available to our core facility. The requested DVRT will be used for imaging cell culture samples where the adherent cells are less than ~50 ¿m thick and have relatively weak fluorescence. For these specimens, the DVRT's iterative constrained deconvolution image restoration surpasses LSCM imaging in resolution and signal to noise. The requested DVRT has shown itself superior for cells susceptible to photodamage or where the fluorescent molecules to be imaged are susceptible to photobleaching or dim, in experiments in which two or more colors of fluorescently labeled molecules are interacting, for acquiring a differential interference contrast (DIC) along with fluorescence and for highly accurate point-visiting for multiple data sets and for 3D and 4D reconstruction. New capabilities provided by the requested DVRT include 3-4 X faster color switching than our current system; 10-20x greater signal-to-noise (S/N) than we can achieve currently with low level fluorescence samples provided by our clients; a controlled environment chamber; an improved extremely accurate XYZ stage with auto-focus to maintain focus within each z-stack (set of optical sections along the Z-axis). This is important for time-lapse studies where stage drift and other mechanical or biological sources cause loss of focus during extended times (hours to days). The requested accessory diode lasers provide the focused illumination needed to perform fluorescence resonance energy transfer (FRET), fluorescence recovery after (laser induced) photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), to study dynamic interactions of specific proteins and subcellular components in living cells. In particular, the DVRT system has proven far superior to anything else available for extremely large files (>2GB) that require 3D or 4D reconstruction in order to properly understand the data. RELEVANCE - The >80 clients in >26 departments of our imaging facility do both basic biomedical and translational medical research. Examples include studies of the survival mechanisms used by disease causing organisms to evade detection in their human hosts, intercellular communication and control in the nervous system, formation of the immunological synapse between lymphocytes and antigen presenting cells, and cell to cell spread by human viruses, and pathogenesis and host-cell survival of Salmonella typhimurium, Francisella tularensis, Neisseria gonorrheae and N. meningitides.