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。在扩展体积上成像这种快速动态对以下方面提出了挑战 标准荧光显微镜。例如，新一代的基因编码电压 其响应时间为毫秒级的指示器正在变得可用。 为了应对这一挑战，我们建议开发一种新型的显微镜，可以进行近- 1 kHz速率高分辨率体积成像，超过1 mm x 1 mm x 0.2 mm尺度。我们提出的解决方案， 称为多Z共焦显微镜，基于两个关键思想。首先，它将高NA检测与 低NA照明。前者导致高信号收集效率;后者导致轴向 在Z深度的扩展范围上扩展照明。第二，它检测到多个信号， 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，等等。以这种方式， 没有信号丢失，信号采集效率保持较高。 我们的显微镜将开发两个版本，基于线扫描和片扫描照明。 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kHz速率），并将设计用于电压成像。相比 传统的线扫描或光片显微镜，我们的线和片的方向平行于光学 轴，而不是垂直于轴。我们的显微镜的两个版本的多功能性将是 增加了光遗传学刺激和组合的共焦反射对比度。 我们得到了阿尔贝托·克鲁兹-马丁博士（波士顿大学，生物学）和薛汉博士（波士顿大学，BME）的帮助，他们 他们都专门从事体内小鼠成像，并在基因或病毒递送新的 探针、动物准备、头部固定、行为方案等。对于电压成像，我们将测试 最先进的指标SomArchon 1（由艾德博伊登博士实验室提供）。的成像能力 1 kHz速率下的大样本量具有普遍适用性，并且可能具有广泛的影响力。我们的目标将 通过对钙离子和钙离子浓度的测量， 在行为正常的小鼠中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.