Deltavision RT Image Restoration System for Established Research Core Facility

描述(由申请人提供)：俄勒冈健康与科学大学的MMI研究核心设施是一个成熟的共享资源设施，为OHSU和波特兰大都市区的26个部门的80多个实验室提供高分辨率荧光显微镜。该应用程序申请资金，将过时的应用精密DeltaVision图像恢复系统替换为新的API DeltaVision RT系统(DVRT)，以及活细胞和激光增强应用所需的附件。DVRT将用于补充我们核心设施可用的激光扫描共焦显微镜(LSCM)的成像。所要求的DVRT将用于对贴壁细胞厚度小于50？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在分辨率和信噪比上都优于LSCM成像。在两种或两种以上颜色的荧光标记分子相互作用的实验中，所要求的DVRT已显示出其对于易受光损伤的细胞或被成像的荧光分子易受光漂白或昏暗的影响的优越性，用于获得与荧光一起的差分干涉对比(DIC)，并且对于多个数据集的高精确点访问以及3D和4D重建。我们要求的DVRT提供的新功能包括：色彩切换速度比当前系统快3-4倍；信噪比(S/N)比我们目前使用客户提供的低水平荧光样本时高10-20倍；环境腔可控；改进的极精确的XYZ工作台，可自动对焦以保持每个Z堆叠内的焦点(沿Z轴的一组光学切片)。这对于延时研究很重要，在延时研究中，阶段漂移和其他机械或生物来源会在延长的时间内(几小时到几天)造成焦点丢失。所要求的辅助二极管激光器提供所需的聚焦照明，以执行荧光共振能量转移(FRET)、(激光诱导)光漂白后的荧光恢复(FRAP)和光漂白中的荧光损失(FLIP)，以研究活细胞中特定蛋白质和亚细胞成分的动态相互作用。特别是，DVRT系统已被证明远远优于任何其他可用于超大文件(&gt；2 GB)的系统，这些文件需要3D或4D重建才能正确理解数据。相关性-我们成像设施的26个部门的&gt；80个客户从事基础生物医学和转化医学研究。例如，研究疾病所使用的生存机制，使生物逃避人类宿主的检测，神经系统中的细胞间通信和控制，淋巴细胞和抗原提呈细胞之间免疫突触的形成，以及人类病毒在细胞间传播，以及鼠伤寒沙门氏菌、图拉氏方济氏菌、淋病奈瑟氏菌和脑膜炎奈瑟菌的致病机制和宿主细胞存活的研究。

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 This application requests funds to replace an outdated Applied Precision DeltaVision image restoration system with a new API DeltaVision RT system (DVRT) with the accessories needed for live-cell and laser enhanced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.