Kilohertz volumetric imaging of neuronal action potentials in awake behaving mice

在神经科学中，长时间动态神经元活动的高速体积成像是一个具有挑战性但基本的目标。神经元活动的传统光学测量大多依赖于钙信号。然而，钙成像只传达了关于神经系统自然信号处理的有限信息，而且它提供的关于大多数神经元中连续发生的抑制和兴奋信号的数据很少或根本没有。相比之下，电压成像可以直接测量神经元的电活动，它有可能克服钙成像固有的局限性。特别是，遗传编码电压指示器(GEVI)的最新进展极大地扩展了电压成像在脑研究中的应用，它们的成功示范反过来又推动了针对电压成像而优化的新型光学仪器的开发。神经元动作电位的光学成像具有挑战性，因为它需要毫秒的时间分辨率。这一要求在三维(3D)成像中变得更加苛刻，在3D成像中，大多数光学成像技术依赖于扫描来获取体积数据，要么像共焦显微镜那样逐点地获取，要么像薄片显微镜那样的平面扫描。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，光场成像可以同时捕获体积数据，使其成为神经元网络3D成像的理想策略。然而，由于光场成像既记录了光线的空间信息，又记录了光线的角度信息，因此通常需要大幅面图像传感器，但由于电子带宽有限，图像传感器的帧速率较低。到目前为止启用的时间分辨率(~数十毫秒)远远不足以解析单个神经元放电事件(~1毫秒)。因此，开发新的成像技术以实现大规模光场数据的高速测量是一项尚未得到满足的需求。这项拟议研究的总体目标是开发一种光场断层扫描显微镜(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体积成像。最近，一种新兴的技术--光场层析成像(LIFT)才使所提出的方法成为可能，该技术在获取用于3D成像的光场数据方面非常高效。Lift不是测量整个光场DataCube，而是只捕获每个透视图像中对象的正面投影，从而显著减少数据负载。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都是1D格式的事实。在拟议的研究中，我们将使Lift适用于3D荧光显微镜。当与GEVI的使用相结合时，合成的系统将为3D神经元网络的高速电压成像提供完整的解决方案。此外，拟议的系统将与行为动物的实验兼容，允许我们将神经元活动与行为联系起来。这样获得的洞察力将有助于在单细胞水平上从电活动解释复杂的动物行为。

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.