Kilohertz volumetric imaging of neuronal action potentials in awake behaving mice

长时间动态神经元活动的高速体积成像是神经科学中一个具有挑战性但又必不可少的目标。传统的神经活动光学测量主要依赖于钙信号。然而，钙成像只能传达神经系统中自然信号处理的有限信息，并且对大多数神经元中持续发生的抑制性和兴奋性信号提供很少或没有数据。相比之下，电压成像可以直接测量神经元的电活动，并且有可能克服钙成像固有的局限性。特别是，基因编码电压指示器（GEVIs）的最新进展极大地扩展了电压成像在大脑研究中的应用，它们的成功演示反过来又推动了开发用于电压成像优化的新型光学仪器。神经元动作电位的光学成像具有挑战性，因为它需要毫秒级的时间分辨率。这一要求在三维（3D）成像中变得更加苛刻，其中大多数光学成像技术依赖于扫描来获取体积数据，无论是像共聚焦显微镜那样的点方向还是像光片显微镜那样的平面。这些系统需要在成像速度和信噪比之间进行权衡。相比之下，光场成像同时捕获体积数据，使其成为神经网络3D成像的理想策略。然而，由于光场成像同时记录光线的空间和角度信息，因此通常需要一个大画幅图像传感器，由于有限的电子带宽，其帧率较低。到目前为止，时间分辨率（约几十毫秒）远远不足以解决单个神经元放电事件（约1毫秒）。因此，开发新的成像技术来实现大规模光场数据的高速测量是一个尚未满足的需求。该研究的总体目标是开发一种光场层析显微镜（LIFT显微镜）方法，用于对清醒行为小鼠的神经元动作电位进行千赫兹体积成像。直到最近，一种新兴技术——光场断层扫描（LIFT）才使该方法成为可能，该技术在获取用于3D成像的光场数据方面效率很高。LIFT不是测量整个光场数据立方体，而是在每个透视图像中仅捕获物体的正面投影，从而大大减少了数据负载。此外，LIFT使用一维（1D）传感器记录数据，利用了大多数快速相机采用1D格式的事实。在拟议的研究中，我们将把LIFT应用于三维荧光显微镜。当与GEVI结合使用时，该系统将为3D神经元网络的高速电压成像提供完整的解决方案。此外，所提出的系统将与有行为的动物实验兼容，使我们能够将神经元活动与行为联系起来。由此获得的见解将有助于从单细胞水平上解释复杂的动物电活动行为。

High-speed volumetric imaging of dynamic neuronal activity over long periods is a challenging but essential goal in neuroscience. Conventional optical measurements of neuronal activity mostly rely on calcium signals. However, calcium imaging conveys only limited information about natural signal processing in the nervous system, and it provides little or no data on the inhibitory and excitatory signals that occur continuously in most neurons. In contrast, voltage imaging allows a direct measure of neuronal electrical activity, and it has the potential to overcome the limitations inherent to calcium imaging. Particularly, the recent advances of genetically encoded voltage indicators (GEVIs) have greatly expanded the use of voltage imaging in brain research, and their successful demonstration has, in turn, motivated developing new optical instrumentation optimized for voltage imaging. Optical imaging of neuronal action potentials is challenging as it requires a millisecond temporal resolution. This requirement becomes more demanding in three-dimensional (3D) imaging, where most optical imaging technologies rely on scanning to acquire volumetric data, either pointwise like confocal microscopy or planes like light-sheet microscopy. These systems suffer from a trade-off between the imaging speed and the signal-to-noise ratio. In contrast, light field imaging captures the volumetric data simultaneously, making it an ideal strategy for 3D imaging of neuronal networks. Nonetheless, because light field imaging records both the spatial and angular information of light rays, it typically requires a large-format image sensor, which has a low frame rate due to limited electronic bandwidth. The temporal resolution enabled so far (~tens of milliseconds) is far from enough to resolve the individual neuron firing event (~one millisecond). Therefore, there is an unmet need to develop new imaging techniques to enable high-speed measurement of large-scale light-field data. The overall goal of the proposed research is to develop a light-field tomographic microscopy (LIFT microscopy) method for kilohertz volumetric imaging of neuronal action potentials in awake behaving mice. The proposed method has been only recently made possible by an emerging technique, light field tomography (LIFT), which is highly efficient in acquiring light field data for 3D imaging. Rather than measuring the entire light field datacube, LIFT captures only an en-face projection of the object in each perspective image, thereby significantly reducing the data load. Furthermore, LIFT records data using one-dimensional (1D) sensors, exploiting the fact that most fast cameras are in 1D format. In the proposed research, we will adapt LIFT for 3D fluorescence microscopy. When combined with the use of GEVI, the resultant system will provide a complete solution to high-speed voltage imaging of 3D neuronal networks. Moreover, the proposed system will be compatible with experiments in behaving animals, allowing us to link neuronal activity to behavior. The insights so obtained will be instrumental to interpreting the complex animal behaviors from the electrical activities at a single-cell level.