In Vivo Imaging Core

双光子激光扫描显微镜是一种可以应用于使用点扫描显微镜和脉冲红外激光对完整组织内的荧光标记细胞进行成像的技术。这种方法可以用于切除的大块组织或活的麻醉动物，这种技术称为活体显微镜（IVM）。为了进行这项技术，所需的设备对除少数调查人员外的所有人来说都是极其昂贵的。耶鲁大学医学院的研究人员很幸运，因为该校已经投资了一台双光子激光器和定制设计的激光扫描显微镜，该显微镜配备了灵敏的外部探测器，可以收集低水平光。该核心C使用的独特显微镜套件允许动物的准备及其随后的成像在相邻的房间中进行。 这一设施通过P30核心赠款得到补贴，并提供给YRDRCC的所有成员。对于这些研究人员中的许多人来说，将这种技术应用于他们的研究极大地加速了他们的研究进展，并以传统体外测定不可能的方式提供了对他们体内生物学问题的实质性见解。与核心PI一起，设施运营商与调查人员进行了协商，以确保实验快速有效地实现。P30的资助使得大量的方法开发成为可能，这些方法实现了许多新的应用，并扩展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会重新出现。例如，维护激光器和显微镜的服务合同的费用将要求大大增加设施使用费，这将造成重大的财务威慑，使许多调查人员，特别是初级调查员无法使用。尽管我们提供了培训和建议，但IVM期间动物麻醉、手术和约束所需的额外技能对大多数研究人员来说似乎令人生畏。因此，本申请建议扩展功能并进一步推广体内成像核心设施。P30资金不仅可以减少这些障碍，还可以获得更多的方法来查询尚未获得的生物事件。拟议的IVICF将对健康和风湿性疾病中体内细胞相互作用、分化和迁移的研究步伐产生巨大影响。

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.