A patterned photostimulation microscope for studying neurons and microcircuitry

描述（由申请人提供）：本申请描述了一种能够图案化激发各种光学敏感目标的多功能显微镜系统。这种定制设计的仪器将用于探索神经细胞生物学，单神经元生理学和微电路功能的前沿问题。它将包含一个脉冲红外光源，用于基于PMT的双光子成像;此外，它将有另一条光路，允许在各种波长（405，473和594 nm）下进行图案化的单光子照明。图案化照明将通过空间光调制器（SLM）技术来实现，该空间光调制器（SLM）技术允许用户定义的照明图案被传递到样本平面（Lutz等人，2008; Nikolenko等人，2008年）。高速CCD摄像机将被用作实验的替代检测器，在实验中，高时间分辨率（即> 1 kHz）的光学测量是必要的。这将允许光学测量许多单个神经元的电活动，这些神经元被一个小组成员开发的新型电压传感器标记。对加州大学洛杉矶分校现有显微镜能力的调查表明，没有具备这些能力的设备;事实上，有两个用户与海外实验室合作，以实现这项工作。 该仪器应允许实验操作几个尖端的光学工具，具有无与伦比的空间和时间精度。其中包括：1）光活化或光转化蛋白质（PA-GFP，Dendra 2，DRONPA）可用于神经系统功能的细胞生物学研究，2）可用于光刺激单个神经元或回路的笼状神经递质，3）光敏通道（通道-和盐视紫红质）或可逆光开关（帕尔斯）以激活或激活特异性电路元件，和4）一种新的，膜电位的超快速光学报告器，能够同时测量许多单个神经元的神经活动。 该应用程序详细说明了仪器系统将如何直接受益于目前资助的10多个NIH项目，其中五个主要用户担任主要研究者。这些实验室的研究旨在了解神经生理学和神经细胞生物学的基本问题，包括兴奋-收缩偶联（Vergara）、胞吐（Schweizer）和神经元可塑性（Martin）中涉及的分子机制，以及脑干呼吸中心（Feldman）、前庭上皮（Schweizer）和小脑皮层（奥蒂斯）中微电路功能的各个方面。 加州大学洛杉矶分校的各个部门和医学院将提供92，922美元的资金，以购买该系统;他们还将每年提供超过46，000美元的持续资金，以支付设备的服务合同。此外，用户组将提供约90，000美元的电生理，微灌注和温度控制设备，以便在受控的生理条件下，神经生理实验可以与光学测量并行进行。这些承诺总额超过25万美元的机构支持证明了加州大学洛杉矶分校对这种仪器的科学热情和强烈需求。 公共卫生相关性：这笔赠款将为一个最先进的显微镜系统提供资金，该系统采用先进的光学技术，能够并行操作和/或测量各种大脑微电路中的神经元活动。该系统还将允许在细胞生物学实验中以前所未有的精度对光学标记的信号蛋白进行复杂的跟踪和操作。加州大学洛杉矶分校有一个大型和高度合作的神经科学研究社区，该设备将加速NIH资助的主要用户实验室的进展，这些用户对呼吸，运动，平衡和学习障碍的基本机制的研究目前由超过15个PHS赠款资助。

DESCRIPTION (provided by applicant): This application describes a multifunctional microscope system capable of patterned excitation of various optically sensitive targets. This custom-designed instrument will be used to explore cutting edge questions in neural cell biology, single neuron physiology, and microcircuit function. It will incorporate a pulsed-IR source for PMT-based two-photon imaging; in addition it will have another optical path allowing for patterned, single-photon illumination at various wavelengths (405, 473 and 594 nm). Patterned illumination will be accomplished with spatial light modulator (SLM) technology which permits user defined patterns of illumination to be delivered to the specimen plane (Lutz et al., 2008; Nikolenko et al., 2008). A high speed CCD camera will be used as an alternative detector for experiments in which high time resolution (i.e.> 1 kHz) optical measurements are necessary. This will allow optical measurements of electrical activity from many single neurons labeled with a novel voltage sensor developed by one of the group members. A survey of existing microscopy capabilities at UCLA demonstrates that no equipment with these capabilities is available; indeed two of the users have collaborations with overseas laboratories to enable the work. This instrument should allow experimental manipulation of several cutting edge optical tools with unparalleled spatial and temporal precision. These include: 1) photoactivatable or photoconvertible proteins (PA-GFP, Dendra2, DRONPA) useful for cell biological studies of nervous system function, 2) caged neurotransmitters that can be used to photostimulate individual neurons or circuits, 3) photosensitive channels (channel- and halorhodopsin) or reversible photo switches (PALs) to activate or inactivate specific circuit elements, and 4) a novel, ultra rapid optical reporter of membrane potential that enables simultaneous measurements of neural activity from many individual neurons. The application details how the instrument system will directly benefit more than 10 currently funded NIH projects on which the five major users serve as Principal Investigators. Research in these laboratories is directed at understanding fundamental issues in neurophysiology and neural cell biology including the molecular mechanisms involved in excitation-contraction coupling (Vergara), exocytosis (Schweizer), and neuronal plasticity (Martin) as well as aspects of microcircuit function in brainstem respiratory centers (Feldman), vestibular epithelium (Schweizer), and in cerebellar cortex (Otis). Various UCLA Departments and the UCLA School of Medicine will provide $92,922 in funding to enable the purchase of the system; they will also contribute more than $46,000 per year in ongoing funding to cover the service contract for the equipment. In addition, the user group will provide approximately $90,000 of electrophysiological, micro perfusion, and temperature control equipment so that neurophysiological experiments can be done in parallel with optical measurements under controlled physiological conditions. These pledges totaling well over $250,000 in institutional support are evidence of the scientific enthusiasm and intense need for this type of instrumentation at UCLA. PUBLIC HEALTH RELEVANCE: This grant would provide funding for a state-of-the-art microscope system incorporating advanced optical technology that enables parallel manipulation and/or measurement of neuronal activity within various brain microcircuits. The system will also permit sophisticated tracking and manipulation of optically-tagged signaling proteins with unprecedented precision in cell biological experiments. UCLA has a large and highly collaborative neuroscience research community and this equipment will accelerate progress in the laboratories of the NIH-funded major users whose research on basic mechanisms underlying disorders of breathing, movement, balance, and learning is currently funded by more than 15 PHS grants.