Fast, large-scale neuronal imaging with multi-z confocal microscopy

摘要 神经元信号可以在毫秒级的时间尺度上变化，通信神经元通常被 几百微米。对扩展卷上如此快速的动态进行映像是一项挑战 标准荧光显微镜。例如，新一代基因编码电压 正在出现响应时间在毫秒量级的指示器。 为了应对这一挑战，我们建议开发一种新型显微镜，它可以执行近- 1 kHz速率高分辨率体积成像，超过1 mm x 1 mm x 0.2 mm比例。我们提出的解决方案， 被称为多Z共焦显微镜，是基于两个关键思想。首先，它将高NA检测与 低噪声照度。前者导致高信号采集效率；后者导致轴向 在Z深度的扩展范围内扩展照明。其次，它会检测到多个信号 使用轴向分布的多个共焦针孔扩展深度范围。针孔是 反射，使得被一个针孔拒绝的信号被发送到下一个针孔，以此类推。以这种方式， 信号不丢失，信号采集效率保持较高。 我们将开发两种版本的显微镜，基于行扫描和片状扫描照明。 前者提供了更好的光学切片，将被设计用于钙成像。后者 提供更高的速度(接近千赫速率)，并将为电压成像而设计。与之形成鲜明对比的是 传统的行扫描或薄片显微镜，我们的线条和薄片是平行于光学的 轴，而不是垂直于轴。我们两个版本的显微镜的多功能性将是 增加了光发生刺激和组合的共焦反射对比度。 我们已经得到了Alberto Cruz-Martin博士(BU，生物学)和薛涵(BU，BME)的帮助，他们 两人都擅长体内小鼠成像，并在遗传或病毒传递新技术方面拥有专业知识 探头、动物准备、头部固定、行为规范等。对于电压成像，我们将测试一个 名为SomArchon1的最先进的指示器(由Ed Boyden博士实验室提供)。想象的能力 在1 kHz频率下的大样本体积具有普遍适用性，并且可以产生广泛的影响。我们的目标是 演示我们的多Z显微镜技术的有效性，通过进行钙离子和 在行为正常的小鼠身上对整个神经元群体进行电压成像。

ABSTRACT Neuronal signals can vary on millisecond timescales, with communicating neurons often separated by hundreds of microns. Imaging such fast dynamics over extended volumes presents a challenge for standard fluorescence microscopes. For example, a new generation of genetically encoded voltage indicators are becoming available whose response times are on the order of milliseconds. To address this challenge, we propose to develop a new type of microscope that can perform near- 1kHz-rate high resolution volumetric imaging over 1mm x 1mm x 0.2mm scales. Our proposed solution, called Multi-Z confocal microscopy, is based on two key ideas. First, it combines high-NA detection with low-NA illumination. The former leads to high signal collection efficiency; the latter leads to axially extended illumination over an extended range of Z depths. Second, it detects multiple signals from this extended depth range using multiple confocal pinholes that are axially distributed. The pinholes are reflecting, so that signal rejected by one pinhole is sent to the next pinhole, and so forth. In this manner, no signal is lost, and signal collection efficiency remains high. Two versions of our microscope will be developed, based on line-scan and sheet-scan illumination. The former provides better optical sectioning and will be designed for calcium imaging. The latter provides much higher speed (near kHz-rate) and will be designed for voltage imaging. In contrast to conventional line-scan or light-sheet microscopes, our lines and sheets are oriented parallel to the optical axis rather than perpendicular to the axis. The versatility of both versions of our microscope will be augmented with the addition of optogenetic stimulation and combined confocal reflectance contrast. We have enlisted the help of Drs. Alberto Cruz-Martin (BU, Biology) and Xue Han (BU, BME), who both specialize in in-vivo mouse imaging and have expertise in the genetic or viral delivery of novel probes, animal preparation, head fixation, behavior protocols, etc.. For voltage imaging, we will test a state-of-the-art indicator called SomArchon1 (provided by the Dr. Ed Boyden lab). The ability to image large sample volumes at 1kHz rates is of general applicability and can be broadly impactful. Our goal will be to demonstrate the effectiveness of our Multi-Z microscopy technique by performing calcium and voltage imaging over entire populations of neurons in behaving mice.