TWO PHOTON LASER SCANNING MICROSCOPE

这项提议的总体目标是在加州大学洛杉矶分校脑研究所的显微成像核心中包括一台双光子激光扫描显微镜(TPLSM)，该显微镜配备用于研究活细胞的生理过程。其目的是补充核心内已有的其他成像技术，如共焦显微镜和电子显微镜。通过在细胞成像系统中利用双光子激发，许多重要的新的实验途径将成为可能，该系统配备了利用电生理学和显微注射进行复杂的细胞操作。TPLSM允许在衍射有限的体积内测量荧光，不需要共焦针孔，并且在焦平面外的光漂白和光损伤最小。这使得双光子激发成为在活细胞中进行测量的理想选择。此外，使用更长波长的照明可以在组织内部进行成像。这项提案中的项目来自具有不同研究兴趣的广泛的神经科学教员。几个主要用户建议测量完整组织内细胞中的离子浓度(钙或锌)。这些切片来自大脑的不同区域(Istvan Mody、Chris Colwell、Michael Levine)、视网膜(Nicholas Brecha)、神经内分泌器官(Nancy Wayne、Jonathan Monck)和骨骼肌纤维(Julio Vergara)。另一位用户将研究吸气调节的突触输入到不同类型神经元的荧光团(杰克·费尔德曼)。一些用户对神经系统发育过程中的组织结构很感兴趣，并建议使用荧光标记(绿色荧光蛋白(GFP)、羧基蓝)来追踪神经投射或突触连接(Anthony Campagnoi，Susana Cohen-Cory，Ellen Carpenter)。两名用户正在追踪转基因小鼠和线虫特定神经元群体中表达的GFP靶向蛋白(Anthony Camagnon i，Alex van der Bliek)。另一位用户建议使用GFP来研究完整胃肠道组织内神经元的受体内化(Catia Sternini)。TPLSM的可获得性将显著增强和扩大这些研究人员的研究方向，他们中的大多数都有NIH资助的正在进行的研究项目。

The overall goal of this proposal is to include within the Microscopic Imaging Core of the UCLA Brain Research Institute a two-photon laser scanning microscope (TPLSM) equipped for investigation of physiological processes in living cells. The intention is to complement other imaging technologies, such as confocal microscopy and electron microscopy, which are already available within the Core. Many important new experimental avenues will be made possible by utilizing two-photon excitation in a cellular imaging system equipped for complex cellular manipulation using electrophysiology and microinjection. The TPLSM allows measurement of fluorescence within a diffraction limited volume, without the need for a confocal pinhole and with minimal photobleaching and photodamage outside the focal plane. This makes two- photon excitation ideal for measurements in living cells. In addition, the use of longer wavelengths for illumination allows imaging deep within tissue. The projects in this proposal come from a wide spectrum of neuroscience faculty with different research interests. Several of the major users propose to measure ion concentrations (Ca2+ or Zn2+) in cells within intact tissue. These include slices from various regions of the brain (Istvan Mody, Chris Colwell, Michael Levine), retina (Nicholas Brecha), neuroendocrine organs (Nancy Wayne, Jonathan Monck), and skeletal muscle fibers (Julio Vergara). Another user will study inspiratory-modulated synaptic inputs to various neuron types with fluorophores (Jack Feldman). Several users are interested in the organization of the nervous system during development and propose to use fluorescent labels (green fluorescent protein (GFP), carboxycyanines) to trace neural projections or synaptic connections (Anthony Campagnoni, Susana Cohen-Cory, Ellen Carpenter). Two users are tracing GFP targeted proteins expressed in specific neuronal populations in transgenic mice and C. elegans (Anthony Campagnoni, Alex van der Bliek). Another user proposes to use GFP to study receptor internalization in neurons within intact gastrointestinal tissue (Catia Sternini). The availability of the TPLSM will significantly enhance and expand the research directions of these investigators, most of whom have ongoing NIH-funded research programs.