Laser System for Optogenetic Stimulation for the subcellular investigation of Neural Networks

光遗传学是基于转基因离子通道和泵的应用，这些离子通道和泵可以被光激活。它被认为是控制和监测单个神经元活动的强大工具，从而研究中枢神经系统中的电路。目前所采用的照明技术，如基于LED的广场照明、多模光纤或激光扫描技术，要么缺乏针对单个细胞的空间选择性，要么存在无法同时刺激多个细胞的缺点。在这个项目中，我们的目标是实现一个通用的时空光刺激系统，用于体内和体外光遗传学的应用。我们将利用快速纯相位空间光调制器来开发波前整形的潜力，以应对光遗传学中的一些主要挑战：具有亚细胞分辨率的单细胞刺激、多个细胞的单独刺激和校正像差。数字激光系统将产生定制的波前。将使用两个波长，一个用于细胞激活(473 nm用于兴奋通道视紫红质-2)，另一个用于抑制(589 nm用于兴奋卤视紫质)。铁电液晶光调制器将作为关键部件，用于投射计算机生成的全息图，以形成结构光场。多个斑点可以在毫秒级的时间尺度上单独产生和调制，同时使用膜片钳和微电极阵列等电生理学技术测量响应。将采用迭代波前校正程序来补偿来自光学系统和组织样本本身的折射率变化的像差，并实现亚细胞分辨率。在光遗传学领域，我们将首次实现快速闭环控制，读取激活信号，并实时计算和显示更新的刺激模式。这需要大约10毫秒的小延迟。激光系统将用于对人类诱导的表达通道视紫红质和卤视紫质的多能干细胞的神经网络进行实验。我们将研究随机神经网络和定向神经网络中的连通性，以研究突触可塑性现象，从短期增强到长期增强。

Optogenetics is based on the application of transgenic ion channels and pumps that can be activated by light. It is regarded a powerful tool to control and monitor the activity of individual neurons and hence to investigate circuits in the central nervous system. Illumination techniques employed so far like LED based widefield illumination, multimode fibers or laser scanning techniques lack either spatial selectivity to address single cells or have the disadvantage that multiple cells cannot be stimulated at the same time.In this project, we aim to realize a versatile spatio-temporal light stimulation system for in vivo as well as in vitro applications in optogenetics. We will exploit the potential of wavefront shaping by means of fast phase-only spatial light modulators to meet some of the main challenges in optogenetics: single cell stimulation with subcellular resolution, individual stimulation of multiple cells and correction of aberrations. The digital laser system will generate tailored wavefronts. Two wavelengths will be used, one for cell activation (473 nm for exciting ChannelRhodopsin-2) and the other for inhibition (589 nm for exciting HaloRhodopsin). Ferroelectric liquid-crystal light modulators will be used as the key components to project computer generated holograms for the formation of a structured light field. Multiple spots can be generated and modulated individually on the millisecond time scale, while measuring the responses with electrophysiological techniques such as patch-clamping and microelectrode arrays. Iterative wavefront correction procedures will be applied to compensate aberrations from the optical system and from refractive index variation of the tissue sample itself and to achieve subcellular resolution. For the first time in optogenetics, we will realize a fast closed-loop control, which reads the activation signals and computes and displays an updated stimulation pattern in real-time. A small latency of about 10 ms is required for this. The laser system will be applied to conduct experiments on neural networks derived from human induced pluripotent stem cells expressing ChannelRhodopsin and Halorhodopsin. We will head for the investigation of connectivity in random and directed neural networks to study synaptic plasticity phenomena, ranging from short-term to long-term potentiation.