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

使用多 Z 共聚焦显微镜进行快速、大规模神经元成像

基本信息

批准号：
10088442
负责人：
Jerome Mertz
金额：
$ 44.1万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10088442
关键词：
3-Dimensional Address Animals Behavior Biology Brain imaging Calcium Cannulas Collection Communities Computer software Confocal Microscopy Data Detection Development Devices Effectiveness Engineering Exhibits Fluorescence Generations Genetic Goals Head Image Lasers Light Lighting Microscope Microscopy Modality Modification Monitor Mus Neurobiology Neurons Optics Penetration Performance Photobleaching Phototoxicity Population Preparation Protocols documentation Reaction Time Research Resolution Sampling Scanning Signal Transduction Speed Spottings Techniques Technology Testing Time Viral base design effectiveness evaluation experience fluorescence microscope improved in vivo in vivo calcium imaging instrument interest lens millimeter millisecond neuroregulation neurotransmission novel optogenetics prototype recruit reflectance confocal microscopy sample fixation spatiotemporal technology development user-friendly voltage

项目摘要

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.

抽象的神经元信号在毫秒时尺度上可能会有所不同，通信神经元通常被分开数百微米。成像如此快速的动态超过扩展量给人带来的挑战标准荧光显微镜。例如，新一代的遗传编码电压指标正在可用，其响应时间在毫秒顺序。为了应对这一挑战，我们建议开发一种新型显微镜，该显微镜可以执行接近 1MM x 1mm x 0.2mm尺度上的1kHz速率高分辨率体积成像。我们提出的解决方案，称为Multi-Z共聚焦显微镜，基于两个关键思想。首先，它结合了高NA检测低NA照明。前者导致高信号收集效率；后者导致轴向在Z深度的扩展范围内扩展照明。其次，它检测到来自此的多个信号使用轴向分布的多个共聚焦针孔的扩展深度范围。针孔是反射，以便将一个针孔拒绝的信号发送到下一个针孔，依此类推。这样，不会丢失信号，并且信号收集效率仍然很高。根据线扫描和扫描扫描照明，将开发两个版本的显微镜。前者提供更好的光学切片，并将设计用于钙成像。后者提供更高的速度（接近KHz速率），并将设计用于电压成像。与常规的线扫描或灯页显微镜，我们的线和板平行于光学轴而不是垂直于轴。这两个版本的显微镜的多功能性将是随着添加光遗传刺激和共聚焦反射率对比度增强。我们已经获得了博士的帮助。 Alberto Cruz-Martin（BU，生物学）和Xue Han（BU，BME），他两者都专门从事体内鼠标成像，并且具有新颖的遗传或病毒递送方面的专业知识探针，动物制备，头部固定，行为方案等。用于电压成像，我们将测试最先进的指标称为Somarchon1（由Ed Boyden博士提供）。图像的能力以1kHz速率以1kHz的速率具有一般适用性，并且可能具有广泛的影响。我们的目标意志通过执行钙和行为小鼠整个神经元种群的电压成像。