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

用于神经网络亚细胞研究的光遗传学刺激激光系统

• 批准号: 402988941
• 负责人: Professor Dr. Volker Busskamp, Ph.D.
• 金额: --
• 依托单位: Universitäts-Augenklinik Bonn
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/402988941?language=en
• 关键词: Laser; System; Optogenetic; Stimulation; subcellular

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.

光遗传学是基于应用转基因离子通道和泵，可以被激活的光。它被认为是控制和监测单个神经元活动的有力工具，因此可以研究中枢神经系统中的回路。目前采用的照明技术，如基于LED的宽场照明，多模光纤或激光扫描技术缺乏空间选择性，以解决单个细胞或有缺点，不能在同一时间刺激多个细胞。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，在体内以及在体外的光遗传学应用。我们将利用快速相位空间光调制器的波前整形潜力，以满足光遗传学中的一些主要挑战：亚细胞分辨率的单细胞刺激，多个细胞的个体刺激和畸变校正。数字激光系统将产生定制的波前。将使用两个波长，一个用于细胞活化（473 nm用于激发卤素视紫红质-2），另一个用于抑制（589 nm用于激发卤素视紫红质）。铁电液晶光调制器将被用作投影计算机生成的全息图以形成结构光场的关键部件。可以在毫秒时间尺度上单独产生和调制多个点，同时使用膜片钳和微电极阵列等电生理技术测量响应。将应用迭代波前校正程序来补偿来自光学系统和来自组织样本本身的折射率变化的像差，并实现亚细胞分辨率。在光遗传学中，我们将首次实现快速闭环控制，该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 ms的小延迟。该激光系统将用于对来自表达视紫红质和盐视紫红质的人类诱导多能干细胞的神经网络进行实验。我们将研究随机和定向神经网络的连通性，以研究突触可塑性现象，从短期到长期增强。