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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.

摘要