TWO PHOTON LASER SCANNING MICROSCOPE

该提案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），用于研究活细胞的生理过程。其目的是补充其他成像技术，如共聚焦显微镜和电子显微镜，这些技术已经在核心中可用。许多重要的新实验途径将成为可能，利用双光子激发在细胞成像系统装备复杂的细胞操作使用电生理和显微注射。TPLSM允许在衍射有限的体积内测量荧光，而不需要共聚焦针孔，并且在焦平面外具有最小的光漂白和光损伤。这使得双光子激发成为测量活细胞的理想方法。此外，使用较长波长的照明可以在组织深处成像。本提案中的项目来自不同研究兴趣的广泛的神经科学教师。几个主要用户建议测量离子浓度（Ca2+或Zn2+）在完整组织内的细胞。这些切片包括来自大脑不同区域（伊斯特万·莫迪、克里斯·科尔威尔、迈克尔·莱文）、视网膜（尼古拉斯·布雷查）、神经内分泌器官（南希·韦恩、乔纳森·蒙克）和骨骼肌纤维（胡里奥·维加拉）的切片。另一名用户将研究用荧光团对各种神经元类型的吸气调制突触输入（Jack Feldman）。一些用户对神经系统在发育过程中的组织感兴趣，并建议使用荧光标记（绿色荧光蛋白（GFP），羧基花青素）来追踪神经投射或突触连接（Anthony Campagnoni, Susana Cohen-Cory, Ellen Carpenter）。两名用户正在追踪转基因小鼠和秀丽隐线虫特定神经元群中表达的GFP靶向蛋白（Anthony Campagnoni, 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中的受体内化（Catia Sternini）。TPLSM的可用性将大大增强和扩展这些研究者的研究方向，他们中的大多数人正在进行nih资助的研究项目。

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.