Two-photon Light Field with Neuro-active Sensing for Fast Volumetric Neural Microcircuit Readout

在我们的每一个感觉、思想、记忆、决定和行动的基础上，都有1000亿个神经元通过每秒数万亿次的电脉冲进行交流。在过去的世纪，神经科学家主要在两个尺度上探索大脑功能，即单个神经元（即，通过用电极刺穿它们）和整个大脑区域的（即用脑电图，EEG和功能性磁共振成像，fMRI）。然而，在这两个尺度之间，围绕神经元如何在网络中相互作用以处理和存储信息，形成记忆并产生动作存在很大的知识差距。在过去的10年里，遗传学家已经开发出了控制和用光读出脑细胞活动的方法。它们可以使神经元对光敏感，从而在用特定波长照射时激活或沉默它们。此外，神经元在活跃时可以“发光”或变得更荧光。这些“光遗传学”工具使得将单神经元特性（即，通过电极研究）与功能在群体水平上的进化（通过功能磁共振成像和脑电图）。为了实现这一目标，光学工程师必须首先克服一个关键挑战：哺乳动物的大脑会严重散射和扭曲光线，导致图像模糊，从而混淆哪个神经元是活跃的。在这里，我们建议克服这一限制，利用“光遗传学”的能力，激活单个神经元与光在快速连续。具体来说，我们将依次激活整个大脑体积中的每个神经元，以确定每个神经元的“签名”;也就是说，它在激活时产生的模糊，扭曲的光模式。然后，我们将使用这些活动特征的集合来快速准确地确定哪个神经元在随后的自发活动中激活以及何时激活。我们将实施这种“收集”的方法与三维（3D）成像策略称为“光场”。传统的成像技术可以在一个平面上捕捉物体的聚焦图像，而“光场”则可以在一个单一的shapshot中捕捉不同角度的视角。因此，“光场”方法使我们能够在整个脑组织体积中同时跟踪神经元活动，而不是在单个平面内。这种新颖的“光场”成像与主动传感的结合将显着提高速度（10倍），我们可以跟踪整个体积中单个神经元的活动。在不久的将来，更快，更灵敏的相机和传感器的发展可以提高我们的仪器的体积捕获率到100倍相比，目前的最先进的。此外，在这里，我们将首次实现“光场成像”在“双光子”模式。“双光子”是一种激发荧光的方法，广泛用于生物医学研究。与以前使用的“光场”的蓝色/绿色波长相比，“双光子”利用远小于蓝色和绿色散射的近红外波长，使研究人员能够在散射组织中进行深层成像。我们新的双光子光场仪器将减少失真，从而使我们能够更深入地成像大脑。通过将靶向神经激活与3D光场成像相结合，我们将克服理解神经元如何在网络中相互作用的关键障碍。有了我们的新仪器，神经科学家们最终将能够收集关于神经元如何协同工作以处理和存储信息、做出决策和实现行动的数据。对这些网络级过程的详细了解将为神经元疾病和障碍（如阿尔茨海默氏症）的新疗法的设计提供信息，其中这些功能受到损害。

Underlying our every sensation, thought, memory, decision and action are 100 billion neurons communicating through trillions of electrical impulses each second. Over the past century, neuroscientists have explored brain function on primarily two scales, that of single neurons (i.e., by impaling them with electrodes) and that of entire brain regions (i.e. with electroencephalogram, EEG, and functional magnetic resonance imaging, fMRI). However, between these two scales lies a large knowledge gap surrounding how neurons interact in networks to process and store information, form memories and generate actions. Over the past 10 years, geneticists have developed methods to control and read out brain cell activity with light. They can render neurons sensitive to light to activate or silence them when illuminated with certain wavelengths. In addition, neurons can be made to "glow" or become more fluorescent when active. These "optogenetic" tools make it possible to connect single-neuron properties (i.e., through electrode studies) with functions evolving on the population level (through fMRI and EEG). To achieve this, optical engineers must first overcome a key challenge: the mammalian brain severely scatters and distorts light, resulting in blurry images and thus confusion about which neuron is active. Here we propose to overcome this limitation by utilizing the "optogenetic" ability to activate individual neurons with light in rapid succession. Specifically, we will activate each neuron throughout a brain volume in turn to determine each one's "signature"; that is, the blurry, distorted light pattern it generates when active. We will then use this collection of activity signatures to rapidly and precisely determine which neuron activates and when during subsequent spontaneous activity. We will implement this "collection" approach with a three-dimensional (3D) imaging strategy called "light field." While traditional imaging captures focused images for objects lying in a single plane, "light field" captures perspectives from different angles within a single shapshot. The "light field" approach thus enables us to track neuronal activity simultaneously throughout a volume a brain tissue rather than within a single plane. This novel combination of "light field" imaging with active sensing will significantly increase the speed (10-fold) with which we can track the activity of single neurons throughout a volume. In the near future, development of faster, more sensitive cameras and sensors could increase our instrument's volume capture rates to 100-fold compared to the current state-of-the-art. Moreover, here we will, for the first time, implement "light field imaging" in "two-photon" mode. "Two-photon" is a method to excite fluorescence that is used widely in biomedical research. In contrast to the blue/green wavelengths previously used with "light field," "two-photon" utilizes near-infrared wavelengths that are far less scattered than blue and green, enabling researchers to image deep in scattering tissues. Our new two-photon light field instrument will decrease distortion and thus enable us to image deeper into the brain.By combining targeted neural activation with 3D light-field imaging, we will overcome a key barrier to understanding how neurons interact in networks. With our new instrument, neuroscientists will at last be able to collect data on how neurons work together to process and store information, make decisions and effectuate actions. A detailed understanding of these network-level processes will inform the design of new therapies for neuronal diseases and disorders, such as Alzheimer's, in which these functions are compromised.