LSM 5 Live Microscope for Cell Imaging Shared Resource

描述（由申请人提供）：我们建议获得新开发的高速激光扫描_狭缝共聚焦显微镜（LSM 5 Live，Zeiss），以支持范德比尔特大学的五名NIH资助的研究人员的研究，这些研究需要对活体样本中的快速动态事件进行成像。校园里没有其他显微镜具有同等的功能，因此，该系统为大量的调查人员提供了巨大的潜力。新仪器将被纳入细胞成像共享资源（CISR）。CISR为大约250个VUMC实验室提供专业的光学显微镜服务，其中大部分是NIH资助的，包括7个研究中心;经过培训的用户可以每周7天，每天24小时访问该设施。CISR拥有12年的卓越服务，可靠性和自给自足的历史;显微镜的使用目前约为9000小时/年。在最近的评估中，我们证实LSM 5 Live可以采集高达120（512 x512）帧/秒的可行共焦图像。我们以100- 250帧/秒的速率对小鼠肌肉的微脉管系统中的流动血细胞进行成像。我们还通过将压电物镜聚焦与高帧率相结合，以3个z系列/秒的速率获得3D图像（20切片/40微米），从而对胚胎斑马鱼跳动的心脏中所有单个心脏细胞的运动进行成像。该仪器的信号收集效率是优秀的，在我们的可行性实验所需的条件下，光漂白是最小的。重要的是，我们可视化了许多生物过程，而没有明显的，显著的光损伤，例如，在> 12，000帧的数据之后，胰岛中的钙振荡是持续的。与点共聚焦激光扫描系统相比，信号漂白比似乎更有利。对所请求的仪器有直接、基本和适当需求的五个主要用户将使用总仪器活动的70%。他们将用它来研究：1）心血管发育和先天性心脏病的细胞和分子机制的斑马鱼模型; 2）C。elegans减数分裂和生殖分子过程模型; 3）完整功能胰岛中的刺激-分泌偶联; 4）燃料代谢及其与糖尿病和心脏病的关系的小鼠模型;和5）新血管形成和移植胰岛功能的小鼠模型。将LSM 5 Live放置在已建立并广泛使用的CISR中，不仅可以推进该提案中主要用户的研究，而且还可以提供重要的机会来推进NIH赞助的研究，让更多的研究人员研究以前无法进行科学观察的生物学和疾病过程的活体模型的特征。

DESCRIPTION (provided by applicant): We propose to acquire a newly developed high-speed laser scanning_slit-confocal microscope (LSM 5 Live, Zeiss), to support research of five NIH-funded investigators at Vanderbilt University that require imaging of rapid dynamic events in live samples. No other microscope on campus has equivalent capabilities, therefore, this system offers significant potential for a large number of investigators. The new instrument will be incorporated into the Cell Imaging Shared Resource (CISR). The CISR provides expert optical microscopy services for about 250 VUMC laboratories, most are NIH-funded, including seven research centers; trained users can access the facility 24 hours/day, 7 days a week. The CISR has a 12-year history of outstanding service, reliability and self-sufficiency; microscope usage is currently about 9000 hours/year. During a recent evaluation we confirmed that the LSM 5 Live can acquire viable confocal images up to 120 (512x512) frames/sec. We imaged flowing blood cells in microvasculature of mouse muscle at rates of 100- 250 frames/sec. We also imaged motions of all individual cardiac cells throughout the beating heart of an embryonic zebrafish by combining piezo-objective focusing with high frame rates to obtain 3-D images (20slices/40microns) at a rate of three z-series/sec. The signal collection efficiency of the instrument was excellent and photobleaching was minimal under the conditions required for our feasibility experiments. Importantly, we visualized numerous biological processes without obvious, significant photo-damage, e.g., calcium oscillations in pancreatic islets were persistent following >12,000 frames of data. The signal-to- bleaching ratio appeared more favorable compared to a point-confocal laser scanning system. Five Major Users with immediate, essential and appropriate need for the requested instrument will utilize 70% of total instrument activity. They will use it to study: 1) zebrafish models of cellular and molecular mechanisms of cardiovascular development and congenital heart disease; 2) C. elegans models of meiosis and molecular processes of reproduction; 3) stimulus-secretion coupling in intact functioning pancreatic islets; 4) mouse models of fuel metabolism and it's relationship to diabetes and heart disease; and 5) mouse models of neo-vascularization and functionality of transplanted islets. Placing an LSM 5 Live in the established and widely used CISR will not only advance the research of the Major Users in this proposal, but also provide significant opportunity to advance the NIH-sponsored research of many more investigators studying features of living models of biology and disease processes previously inaccessible to scientific observation.