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最先进的商业多光子显微镜，专门配置和定位，以适应一系列翻译成像方法，主要用于研究人类正常和病变活检组织和器官切片中的细胞-细胞动力学。该仪器将专门针对人体组织/BSL-2进行配置和认证，并将位于UCSF帕纳萨斯高地生物成像发展中心（BIDC）的开发空间，毗邻UCSF医院。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技术的设施提供急需的能力，并将促进该技术向目前因仪器可用性和配置而被边缘化的有前途的领域的过渡。该仪器将主要由11名美国国立卫生研究院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将促进15个美国国立卫生研究院资助的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延时成像，通常用荧光染料偶联的非刺激性抗体或蛋白质偶联物显示，直接应用于组织活检或多余的移植组织。它还将适应在比较小鼠模型中各种荧光蛋白的成像。它位于BIDC，在该提案中，大约80%的调查人员使用了它，该设施所服务的大型校园社区也通常可以访问它。我们要求的系统，蔡司710LM在a - b试验中被选中，比较我们定制的显微镜，其设计已被证明与深层组织/高灵敏度数据收集兼容，与商业产品。该显微镜将配备一个环境室，以及用于舞台器官培养的模块，一个电动舞台，以及用于高分辨率和高灵敏度成像的高效pmt。为了在3D环境中以最高的光学分辨率从活细胞或组织中获得最佳的数据收集，需要浸入水浸泡物镜。高灵敏度PMT和32元素“光谱”PMT检测阵列的组合被要求允许成像所有常用的染料组合和/或荧光蛋白变体，但也扩大局部成功使用多通道自身荧光背景减法来增强弱信号。加州大学旧金山分校的公共用户群有两个功能强大的定制多光子仪器和两个旧的商业改造。然而，由于正在运行的典型实验的高容量和长时间，功能仪器被超额订购。此外，“平移”成像需要一种显微镜，该显微镜既要经过人体研究的专门认证，又要有足够的能力适应人体组织样本的短交付时间。

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.