In Vivo Imaging Core

双光子激光扫描显微镜是一种利用点扫描显微镜和脉冲红外线激光对完整组织内带有荧光标记的细胞进行成像的技术。这种方法既可以用于切除的大块组织，也可以用于活体麻醉的动物，这项技术被称为活体显微镜(IVM)。为了执行这项技术，需要的设备对除了少数研究人员之外的所有人来说都是昂贵的。耶鲁大学医学院的研究人员是幸运的，因为该学院已经投资了一种双光子激光和定制设计的激光扫描显微镜，配备了灵敏的外部探测器，允许收集低水平的光。这套由Core C使用的独特显微镜套件允许在相邻的房间内进行动物的准备和随后的成像。 这一机制已通过P30核心赠款得到补贴，并向青年会的所有成员提供。对于这些研究人员中的许多人来说，将这项技术应用于他们的研究大大加快了他们的研究进度，并以一种传统的体外分析无法实现的方式，为他们在体内的生物学问题提供了实质性的见解。与核心PI一起，设施运营商与调查人员进行了磋商，以确保实验快速有效地取得成果。P30的资助使实质性的方法开发成为可能，使许多新的应用成为可能，并扩大了可以成像的荧光团组合和组织类型。 然而，如果没有额外的P30核心资金，许多阻碍研究人员将这种方法应用于体内研究的障碍将重新浮出水面。例如，维护激光和显微镜的服务合同的费用将要求大大增加设施使用费，这将构成重大的财务威慑，使许多调查人员，特别是初级私人投资主任无法使用激光和显微镜。尽管我们已经提供了培训和建议，但在IVM过程中，动物麻醉、手术和约束所需的额外技能似乎令大多数研究人员望而却步。因此，本申请建议扩展能力，进一步促进体内成像核心设施。P30资助不仅将减少这些障碍，而且还将获得更多的方法来查询尚未获得的生物事件。拟议的体外受精将对健康和风湿病体内细胞相互作用、分化和迁移的研究速度产生巨大影响。

Two-photon laser scanning microscopy is a technique that can be applied to the imaging of fluorescently tagged cells within intact tissue using point scanning microscopes and pulsed infra-red lasers. This approach can be employed with either excised bulk tissue or with live, anesthetized animals, a technique referred to as intravital microscopy (IVM). In order to perform this technique, equipment is required that is prohibitively expensive to all but a few investigators. Yale University School of Medicine investigators are fortunate in that the school has already invested in a two-photon laser and custom designed laser scanning microscope equipped with sensitive external detectors that allow for the collection of low level light. A unique microscopy suite for use by this Core C permits the preparation of animals and their subsequent imaging to occur in adjacent rooms. This facility has been subsidized through a P30 Core grant and made available to all members of the YRDRCC. For a multitude of these investigators, the application of this techniques to their research has greatly accelerated the progress of their research and provided substantial insights into their in vivo biological questions in a way that is not possible with traditional in vitro assays. Together with the Core PI, the facility operator has consulted with investigators to ensure that experimentation came to fruition quickly and efficiently. P30 funding has made possible substantial methods development that enabled many novel applications, and expanded the fluorophore combinations and tissue types that could be imaged. Nevertheless, without additional P30 Core funding, many barriers that prevent investigators from applying this approach to their in vivo research would resurface. For example, the cost of the service contracts for maintenance of the laser and microscope would require that facility user fees be greatly increased, posing a significant financial deterrent that would prohibit its use by many investigators, especially junior PIs. The additional skills needed for the animal anesthesia, surgery and restraint during IVM appear daunting to most researchers, despite the training and advice that we have made available. Therefore, this application proposes to expand the capabilities and further promote the In Vivo Imaging Core Facility. P30 funding would not only reduce these barriers but also acquire additional methodologies to query as yet inaccessible biological events. The proposed IVICF would have a dramatic impact on the pace of research into cell-interaction, differentiation and migration in vivo in both health and in rheumatic diseases.