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

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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.