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完善的共享资源设施，为OHSU和波特兰都会区26个部门的80多个实验室提供高分辨率荧光显微镜。该申请要求资金用新的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附件。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将用于成像细胞培养样品，其中贴壁细胞小于~50¿m厚，具有相对较弱的荧光。对于这些样本，DVRT的迭代约束反卷积图像恢复在分辨率和信噪比方面优于LSCM成像。在两种或多种荧光标记分子相互作用的实验中，对于易受光损伤的细胞或要成像的荧光分子易受光漂白或暗淡的细胞，所要求的DVRT已显示出其优越性，用于获得微分干涉对比度（DIC）和荧光，以及对多个数据集和3D和4D重建进行高精度的点访问。所要求的DVRT提供的新功能包括比我们当前的系统快3-4倍的颜色切换速度；比我们目前使用客户提供的低水平荧光样品所能达到的信噪比（S/N）高10-20倍；受控环境室；一个改进的极其精确的XYZ舞台与自动对焦，以保持焦点在每个z-堆栈（一组沿z轴的光学部分）。这对于延时研究很重要，因为阶段漂移和其他机械或生物来源会导致在较长时间内（数小时到数天）失去焦点。所要求的附属二极管激光器提供荧光共振能量转移（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）所需的聚焦照明，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，对于需要3D或4D重建才能正确理解数据的超大文件（>2GB）， DVRT系统已被证明远远优于其他任何可用的系统。相关性-我们的成像设施有26个部门的80个客户从事基础生物医学和转化医学研究。例如，研究致病生物逃避人类宿主检测的生存机制、神经系统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、鼠伤寒沙门氏菌、土拉弗朗西斯菌、淋病奈瑟菌和脑膜炎奈瑟菌的发病机制和宿主细胞存活。

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 This application requests funds to replace an outdated Applied Precision DeltaVision image restoration system with a new API DeltaVision RT system (DVRT) with the accessories needed for live-cell and laser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