Multiphoton Instrumentation for Translational Assays from Human Tissue Biopsies

描述（由申请人提供）：我们正在申请资金购买一个国家的最先进的商业多光子显微镜，专门配置和定位，以适应平移成像方法的投资组合，主要致力于追求在人类正常和患病的活检组织和器官切片的细胞-细胞动力学的研究。该仪器将针对人体组织/BSL-2进行专门配置和认证，并将位于UCSF Parnassus Heights生物成像开发中心（BIDC）的开发空间，毗邻UCSF医院。正如提案中所述，该应用程序代表了临床和基础科学家的专业知识合作。这将通过一个精通该领域的设施提供急需的能力，并将促进将该技术过渡到目前因仪器可用性和配置而被边缘化的有前途的领域。该仪器将主要由11名NIH资助的研究人员及其实验室核心使用，这些研究人员和实验室促进了15个NIH资助的R- P-或U-级项目。该显微镜系统将用于的主要实验类型包括活组织中细胞的多维时移成像，通常用荧光染料缀合的非刺激性抗体或蛋白质缀合物显示，直接应用于组织活检或多余的移植组织。它还将适应比较小鼠模型中各种荧光蛋白的成像。该设施位于BIDC内，研究人员对此提案的利用率约为80%，该设施所服务的较大校园社区也将普遍使用该设施。我们所要求的系统，蔡司710 LM是在A-B试验中选择的，该试验比较了我们定制的显微镜，其设计已被证明与深层组织/高灵敏度数据收集兼容，并与商业产品进行了比较。该显微镜将配备一个环境室以及用于台上器官培养的模块、一个电动载物台和用于高分辨率和高灵敏度成像的高效PMT。为了以最高的可实现的光学分辨率从3D环境中的活细胞或组织中最佳地收集数据，需要浸渍水浸没物镜。要求高灵敏度PMT和32元件“光谱”PMT检测阵列的组合，以允许所有常用的染料组合和/或荧光蛋白变体的成像，而且还使用多通道自体荧光背景扣除方法来增强较弱的信号，以扩展局部成功。加州大学旧金山分校的公共用户群有两个高功能的定制多光子仪器和两个较旧的商业适配。然而，功能性仪器由于运行的典型实验的高容量和长时间流逝而被超额订购。此外，“平移”成像需要一个显微镜，这是专门为人类研究认证，并有足够的能力，以适应人类组织样本的短交货时间。

DESCRIPTION (provided by applicant): We are requesting funds to purchase a state-of-the-art commercial multiphoton microscope specifically configured and situated to accommodate a portfolio of translational imaging approaches, largely dedicated to pursuing studies of cell-cell dynamics in human normal and diseased biopsy tissues and organ slices. The instrument will be specifically configured and certified for human tissues/BSL-2 and will be located in the UCSF Parnassus Heights Biological Imaging Development Center's (BIDC) development space, adjacent to the UCSF hospital. This application represents a collaboration of expertise from clinical and basic scientists, as described in the proposal. This will provided much-needed capacity through a facility well-versed in the art and will facilitate the transition of the technology into promising areas that are currently marginalized by instrument availability and configuration. The instrument will be primarily used by a core of 11 NIH funded investigators and their laboratories which facilitate 15 NIH-funded R- P- or U- level projects. The principal type of experiment for which this microscope system will be used include multidimensional timelapse imaging of cells in living tissues, typically revealed with fluorochrome conjugated non-stimulatory antibodies or protein conjugates, directly applied to tissue biopsies or to redundant transplant tissues. It will also accommodate imaging of a wide variety of fluorescent proteins in comparative mouse models. Located in the BIDC and utilized approximately 80% by the investigators on this proposal, it will also be generally accessible by the larger campus community served by this facility. The system we are requesting, a Zeiss 710LM was chosen amongst A-B trials comparing our custom built microscopes, whose design has proven compatible with deep tissue/high sensitivity data collection, with the commercial offerings. The microscope will be equipped with an environmental chamber as well as modules for on-stage organ culture, a motorized stage, and high-efficiency PMTs for high resolution and high sensitivity imaging. Dipping water immersion objectives are requested for the optimal collection of data from live cells or tissues in a 3D environment at the highest achievable optical resolution. A combination of high-sensitivity PMTs and 32-element 'spectral' PMT detection arrays is requested to permit imaging of all commonly used dye combinations and/or fluorescent protein variants but also to extend local success using multichannel autofluorescence background subtraction methods to enhance weaker signals. The public userbase at UCSF has two highly functional custom-built multiphoton instruments and two older commercial adaptations. However, the functional instruments are oversubscribed due to the high volume and long timelapses of the typical experiments being run. In addition, 'translational' imaging requires a microscope which is both specifically certified for human studies and which has sufficient capacity to accommodate the short lead-times of human tissue samples.