【作者】 刘亚丰；

【导师】 曾绍群；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2012， 博士

【摘要】 多细胞或多神经突起的选择性激活对于研究神经元整合、神经元计算非常重要。近几年，基因工程与光刺激技术结合，推动了一项新技术光遗传技术的诞生，它可以快速、无损、精确地激活表达光敏感通道的细胞，进而选择性地控制特定神经元或局部神经环路的活动。目前荧光显微镜使用的光源大都是宽场光源，进行光刺激时会把整个视场中的所有细胞或整个神经环路激活。激光扫描显微镜通过扫描镜实现多细胞的选择性激活，但由于扫描镜内在的固有惯性，限制了扫描速度和多个位置的选取。最近，空间光调制器被引入显微镜系统中实现了无惯性的选择性激活，但其较低的刷新频率和衍射效率限制了该技术的应用。相比而言，声光偏转器具有随机寻址能力，能够快速无惯性引导激光到任意位置，良好的空间选择性使得声光偏转器非常适合多细胞选择性激活。为了实现以上目的，本文首先利用声光偏转器作为光束扫描器件，结合现代光学显微镜技术，建立了独立的随机光刺激系统，相关的工作主要有：（1）在光路结构设计中，采用二维声光偏转器实现激光光束的二维扫描，并采用通用光学器件、光学调节架实现与显微镜系统耦合，实现生物样品显微放大、可视化处理，引导激光刺激期望的细胞或神经元；在机械设计中，采用模块化设计思想，使得系统结构紧凑，方便与各种商用显微镜系统耦合；为控制声光偏转器工作，设计了驱动电路，它包括标准的数据采集卡、开关电源和控制电路，用于数据传输和与其它外设通信；基于LabVIEW应用软件，开发一套独立的应用控制软件，拥有多种刺激模式，软件可读性好，操作方便。（2）测试了随机光刺激系统的工作性能，单点最快时间可以达到10μs。在40倍物镜下，刺激光斑为1.38μm，刺激范围为164μm。光刺激功率波动<5%，刺激定位误差小于1%。实验结果证明系统具备多点快速激活的能力，系统工作稳定，具有很好的重复性。其次，为了分析测试系统的激活模式与激活效果以及开展生物学应用，与上海神经所的章晓辉研究员、王佐仁研究员两课题组展开合作研究，主要的工作有：（1）利用表达有光敏感通道的HEK293细胞和海马神经元，证明系统在~10ms内可以激活细胞和诱导出动作电位。提出刺激光功率决定系统刺激的空间分辨率，并证明在一定的功率条件下，系统能够激活单细胞而不会激活周围其它细胞，显示系统具备局部激活能力，而采用双光子激发将能够提高系统的空间分辨率，便于实现单细胞激活。分析了点刺激模式与区域刺激模式的不同，提出采用区域刺激模式可以显著提高去极化峰值电流、动作电位的诱导频率及诱导成功率。提出对于区域刺激模式，采用随机扫描方式比顺序扫描方式和环形扫描方式更有效激活细胞。随机光刺激系统能够在微秒水平控制激光刺激多个位置，刺激时间、刺激间隔、刺激位置、刺激强度等可精准调控，是一种超灵活、多模式调控细胞或神经元的工具。（2）利用随机光刺激系统，对小鼠体感皮层神经环路和果蝇嗅觉系统进行了光刺激试验，证明系统适用于不同尺寸细胞的光刺激，系统可以选择性激活多个细胞，可以模拟神经元多个信号输入，进行神经元运算研究和信息整合研究，有利于揭示单个神经元和特定神经网络的功能，证明随机光刺激系统对于今后的神经科学研究具有重要的应用价值。更多还原

【Abstract】 The combination of genetic engineering and photostimulation has boosted anemergence of a new technology–optogenetics, which provides a fast, non-invasive, andselective control of neuronal activity. However, the normal wide-field light illuminationused in the most fluorescence microscopes results in Optogenetic activation of all the cellsor the whole neural circuits in the field of view (FOV). A laser scanning microscopeequipped with galvanometer-driven scanning mirrors has been oftern used to achieveselective Optogenetic activation of multiple cells. Nevertheless, the scanning speed andthe targeting positions are limited because of the inherent inertia of the scanning mirrors.Recently, the spatial light modulator (SLM) was introduced into the microscope to achieveselective activation in a non-inertial way. However, the relatively lower refresh rate andlower diffraction efficiency of SLM have limited the application of this technology. Incontrast, acousto-optical deflector (AOD) with the advantage of fast random addressinghas been used to deliver laser non-inertia to multiple pre-selected sites for light activationof caged compounds in cultured neurons. The excellent spatial selection also makes AODsuitable for selectively stimulation of multiple cells or neurons that express light-sensitivechannels.In the first part of present study, a random-access photostimulation system forselective stimulation based on a pair of perpendicularly oriented AODs has beendeveloped in order to achieve much faster laser activation among multiple positions.Related works are summarized as follows:(1) To achive the scanning of laser beam, a pair of AODs were orientedperpendicularly in the laser light path. The scanning beam was focused onto samples byobjective for the light stimulation. General optical devices and adjusting brackets wereused in the system to couple with a commercial microscope for observing the samples andselecting interested cells or neurons. Mechanical parts of this system were designed ccording to the concept of modular design, which made all optical parts and electroniccomponents were encapsulated into a small box. This complete all-in-a-box allows thissystem easily attached to different brands of commercial microscopes. Meanwhile, anAOD controller box has been built with a standard data sampling card, electronic circuitsfor data transmission and communication with computer and other peripherals. Oneapplication routine was developed using LabVIEW software for controlling thephotostimulation process and the codes of the program is accessibly by the end users.(2) The performances of this system were then tested. It possesses a highrandom-addressing rate up to10μs per site. The spatial resolution and stimulation rangeof the system are1.38and164μm respectively, under40×objective. The fluctuation oflaser power in this system is less than5%, and spatial targeting error is less than1%.These assessment data indicate that the system is applicable for rapid and stable laserstimulation of mutltiple postions.In the second part of this study, we further analysed the performances of laeractrivation of light-sensitive channels in cultures, brain slices or intact Drosophila brain,collaborating with groups of Drs. Zhang Xiaohui and Wang Zuoren at the Institute ofNeuroscience, Chines Academy of Sciences. The results are summarized as follows:(1) To evaluate the efficacy of stimulating cells, light-sensitive channels ChIEFs wereexpressed in HEK293cells and hippocampal neurons in cultures. Our results show thatthis system is able to activate cells or induce a neuron to fire action potentials in10milliseconds stimulation. The spatial resolution of this system was correlated with laserpower and could achieve single-cell activation by using relatively low laser power. Inaddition, the spatial resolution can be substantially improved equipped with two-photonlaser. Region stimulation mode could increase the amplitude and the slope of inducedcurrent, and evoke much high-frequency action potentials after spot stimulation mode andregion stimulation mode were analysized in detail. The random scanning way in regionmode could activate cells more effectively than the two other. The ultra-fast multi-site stimulation features of the system provide a flexible solution for manipulating neuronalactivity at high efficiency and multiple modes.(2) Finally, we demonstrated the versatility of this high-resolution system indissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo.These data indicate that this system could be applied to varied species with different cellsize for mapping the synaptic connectivity and studying the neuronal informationprocessing. These results prove this system will be widely used in neuroscience.更多还原