Intravital Microscope with Holographic Photostimulator

我们对大脑功能的理解有赖于在不同的方法论水平上获得知识。记录神经元的活动可以为神经元可能参与的大脑功能提供信息。然而，为了机械地描述大脑功能，神经元活动需要以一种因果的方式与大脑功能和行为联系起来。在单细胞或网络水平上操纵神经元活动构成了实现这一目标的重要方法学途径，并可通过光遗传学实现。为了以高空间和时间精度激活光遗传神经元，刺激光需要用专用的光刺激器雕刻。在这里，我们请求资金建立一个显微镜装置，使这种实验成为可能，并由一个双光子显微镜和一个全息光刺激器组成。在这个系统中，空间光调制器(SLM)被用来在神经组织内产生三维光掩模(又名全息图)。这些全息图是通过预定义的SLM刺激光的干涉图案产生的，并实现了精确的空间控制，也是沿着光轴。与该装置的成像臂相结合，可以同时测量(钙成像)和操作(光遗传全息)神经元活动。对设置的进一步硬件和软件添加允许同时呈现视觉刺激和记录额外的实验参数(例如，活体中的眼睛位置)。为了在图宾根大学建立这项技术，我们需要资金来建立一个集成的全息显微镜装置(没有激光)。我们计划在斑马鱼的视觉运动系统(体内)、小鼠视网膜的视觉功能(体外)以及睡眠小鼠的记忆巩固(体内)的几个项目中使用这种设置。一个例子项目将利用斑马鱼的动眼神经后脑，研究持续活跃的经常性网络中的信息存储机制。脊椎动物的后脑包含速度-位置神经积分器，通过其持续的活动存储关于当前眼睛位置的信息。利用全息光刺激，我们将操纵功能识别的整合神经元的活动，并观察和解释由此导致的眼睛位置和网络活动的变化。这些实验将展示积分器的功能结构，并揭示这个持续活跃的网络的信息存储机制。

Our understanding of brain function hinges on the gain of knowledge at different methodological levels. Recording of neuronal activity can inform about the brain functions a neuron is likely involved with. However, to mechanistically describe brain function, neuronal activity needs to be linked to brain function and behavior in a causal manner. Manipulation of neuronal activity at single cell or network level constitutes an important methodological approach for this goal and can be realized via optogenetics. To optogenetically activate neurons with high spatial and temporal precision, the stimulation light needs to be sculpted with dedicated optical stimulators. Here, we request funds for a microscope setup that enables such experiments and consists of a two-photon microscope and a holographic photostimulator. In this system, a spatial light modulator (SLM) is used to generate three-dimensional photo-masks (aka holograms) within the nervous tissue. These holograms are evoked via pre-defined interference patterns of the SLM stimulation light and enable precise spatial control, also along the optical axis. In combination with the imaging arm of the setup, neuronal activity can be measured (calcium imaging) and manipulated (optogenetic holography) at the same time. Further hardware and software additions to the setup allow for simultaneous visual stimulus presentation and recording of additional experimental parameters (e.g. eye positions in vivo). To establish this technology at the University of Tübingen, we need funds for an integrated holographic microscopy setup (without laser). We plan to use this setup in several projects on the visuomotor system in zebrafish (in vivo), visual function in the mouse retina (ex vivo), as well as memory consolidation in sleeping mice (in vivo). One example project will investigate information storage mechanisms in persistently active, recurrent networks, making use of the zebrafish oculomotor hindbrain. The vertebrate hindbrain contains the velocity-to-position neural integrator, which stores information about the current eye position via its persistent activity. Using holographic photostimulation, we will manipulate the activity of functionally identified integrator neurons and observe and interpret the resulting changes in eye position and network activity. These experiments will demonstrate the functional structure of the integrator and reveal the mechanisms of information storage of this persistently active network.