LSM 5 Live Microscope for Cell Imaging Shared Resource

描述(由申请人提供)：我们建议购买一台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由NIH资助的五名研究人员的研究，这些研究需要在现场样本中对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能力，因此，该系统为大量研究人员提供了巨大的潜力。新仪器将并入细胞成像共享资源(CISR)。该中心为大约250个VUMC实验室提供专家光学显微镜服务，其中大多数是由美国国立卫生研究院资助的，包括7个研究中心；训练有素的用户可以一周7天、每天24小时访问该设施。在出色的服务、可靠性和自给自足方面，该中心已有12年的历史；目前显微镜的使用时间约为9000小时/年。在最近的一次评估中，我们确认LSM 5 Live可以获得高达120(512x512)帧/秒的可行共焦图像。我们以100-250帧/秒的速度对小鼠肌肉微血管中流动的血细胞进行了成像。我们还通过将压电目标聚焦与高帧频相结合，以三个z系列/秒的速率获得3-D图像(20切片/40微米)，从而对斑马鱼胚胎心脏跳动过程中所有单个心肌细胞的运动进行成像。在可行性实验所要求的条件下，该仪器的信号采集效率很高，光漂白最小。重要的是，我们可视化了许多生物过程，但没有明显的、显著的光损伤，例如，在12,000帧数据之后，胰岛中的钙振荡持续存在。与点共聚焦激光扫描系统相比，信号漂白比似乎更有利。对所要求的仪器有直接、必要和适当需求的五个主要用户将利用仪器活动总量的70%。他们将利用它来研究：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)线虫减数分裂和分子生殖过程的模型；3)完整功能胰岛中刺激-分泌耦合的模型；4)燃料代谢及其与糖尿病和心脏病的关系的小鼠模型；以及5)移植胰岛的新生血管和功能的小鼠模型。将LSM 5 Live放置在已建立和广泛使用的CISR中，不仅将促进该提案中主要用户的研究，而且还将提供重要的机会，推动NIH赞助的更多研究人员研究以前无法进行科学观察的生物和疾病过程的活模型的特征。

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.