A STEM Microscope for High-speed 2-photon Calcium Imaging

用于高速 2 光子钙成像的 STEM 显微镜

基本信息

批准号：
7811542
负责人：
Carlos Portera-Cailliau
金额：
$ 49.89万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7811542
关键词：
3-Dimensional Action Potentials Address Affect American Area Arts Autistic Disorder Behavior Bipolar Disorder Bite Brain Calcium California Cells Cognition Collaborations Color Complement Complex Creativeness Custom Data Detection Development Disease Dyes Economic Recession Electrodes Emotions Epilepsy Fluorescence Foundations Functional Imaging Funding Goals Grant Health Image Imagery Interdisciplinary Study Knowledge Lasers Lead Learning Location Measures Memory Mental Retardation Methods Microscope Microscopy Movement Mus Neocortex Neurons Neurosciences Occupations Optics Organism Pattern Penetration Perception Photons Physiologic pulse Postdoctoral Fellow Recovery Research Resolution Risk Sampling Scanning Schizophrenia Security Sensory Series Signal Transduction Solutions Speed Stream Structure Students Symptoms System Technology Testing Time Tissues Training Translating Vibrissae Wages barrel cortex behavior influence calcium indicator cohort design digital fluorescence imaging fluorophore graduate student improved in vivo innovation instrument minimally invasive neocortical neuronal patterning neuropsychiatry public health relevance relating to nervous system research study response spatiotemporal tool

项目摘要

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) "Enabling Technologies" and specific challenge topic 06-AG-101* Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. It also applies to specific challenge topics 06-NS-101 (Developing minimally invasive measures of neural activity) and 06-NS-103 (Breakthrough technologies for neuroscience). One of the greatest challenges for neuroscience in the 21st century is to understand how the billions of neurons that form the brain communicate with one another to produce complex behaviors. The ultimate benefit from this type of research will come from deciphering how dysfunctional patterns of activity amongst neurons lead to devastating symptoms in a variety of neuropsychiatric disorders. Unfortunately, little is known regarding how neural computations in the brain interpret sensory inputs or generate behaviorally relevant responses. This is due in part to the current lack of tools to interrogate the activity of large numbers of neurons in the intact brain. Through an interdisciplinary research collaboration between physicists and neuroscientists, we have developed a high-speed 2-photon microscope for calcium imaging that combines fast resonant scanning mirrors and multi-beam imaging to achieve image acquisition rates more than 2 orders of magnitude faster than conventional 2-photon microscopes. To avoid the fundamental limitation of scattering ambiguity with multiple beams in deep-tissue 2-photon microscopy, we propose an innovative approach to detect and resolve scattered fluorescence emission from separate beams at different times. Specifically, we split the laser beam into four beam lets and then delay each beam optically from the others by 3 ns. We call this method Spatio- Temporal Excitation-emission Multiplexing (STEM). The signals from all four beams are detected by a state- of-the-art GHz bandwidth photodetector. Our microscope therefore preserves the unique advantages of 2- photon microscopy, including its ability to excite fluorophores deeper in the tissue, its reduced photo damage and its exquisite spatial resolution. We now propose to systematically optimize our STEM microscope in order to achieve fluorescence lifetime imaging (FLIM) capability and 4-color imaging. The ultimate goal is to achieve unprecedented 6-D (x, y, z, t, ¿, ") bio-imaging at the single cell level. In addition, we propose a series of in vivo calcium experiments to systematically dissect the micro-scale connectivity of neocortical circuits. First, we will calibrate our STEM system to demonstrate its superior action potential detection compared to conventional 2-photon calcium imaging. Next, we will examine the spatiotemporal dynamics of large ensembles of layer 2 and layer 3 neurons in barrel cortex in response to whisker deflections, by recording from hundreds of these neurons simultaneously in 2-D and 3-D at unprecedented speeds. Within 2 years, the instrument will be optimized and we will be able to characterize, for the first time, the functional wiring diagram of entire complement of neurons within a volume of neocortex. PUBLIC HEALTH RELEVANCE: We have recently developed a high-speed microscope to record the activity of neurons in the intact brain non-invasively. The goal of the proposed challenge grant is to optimize this instrument and then use it to investigate how brain circuits are assembled during development in areas important for emotion, cognition and creativity, as well as for learning and memory. This innovative tool will allow neuroscientists to design experiments that can generate new ideas regarding how subtle alterations in brain wiring could result in devastating neuropsychiatric disorders such as schizophrenia, autism, mental retardation or bipolar disorder.

描述（由申请人提供）：本申请涉及广泛的挑战领域 (06)“使能技术”和特定挑战主题 06-AG-101* 神经科学蓝图：开发直接评估神经活动的非侵入性成像方法或技术。适用于特定挑战主题 06-NS-101（开发神经活动的微创测量）和 06-NS-103（神经科学的突破性技术）。 21 世纪的神经科学的目标是了解构成大脑的数十亿个神经元如何相互通信以产生复杂的行为。此类研究的最终好处将来自于破译神经元之间功能失调的活动模式如何导致破坏性症状。不幸的是，我们对大脑中的神经计算如何解释行为感觉输入或产生相关反应知之甚少，部分原因是目前缺乏询问完整神经元活动的工具。通过物理学家和神经科学家之间的跨学科研究合作，我们开发了一种用于钙成像的高速 2 光子显微镜，它结合了快速共振扫描镜和多光束成像，可实现比大脑快 2 个数量级的图像采集速率。为了避免深层组织 2 光子显微镜中多光束散射模糊的根本限制，我们提出了一种创新方法来检测和解析来自单独的散射荧光发射。具体来说，我们将激光束分成四个光束，然后将每个光束与其他光束延迟 3 ns，我们将来自所有四个光束的信号称为时空激发发射复用 (STEM)。因此，我们的显微镜保留了 2 光子显微镜的独特优势，包括其激发组织更深处的荧光团的能力，以及其减少的能力。我们现在建议系统地优化我们的 STEM 显微镜，以实现荧光寿命成像 (FLIM) 能力和 4 色成像。 , t, ¿ , ") 单细胞水平的生物成像。此外，我们提出了一系列体内钙实验来系统地剖析新皮质回路的微观连接。首先，我们将校准我们的 STEM 系统以展示其卓越的动作潜力接下来，我们将通过同时记录数百个这样的神经元来检查桶状皮层中第 2 层和第 3 层神经元响应晶须偏转的时空动力学。两年内，该仪器将以前所未有的速度进行优化，我们将能够首次表征新皮质体积内整个神经元的功能接线图。公共健康相关性：我们最近开发了一种高速显微镜，可以无创地记录完整大脑中神经元的活动。拟议的挑战拨款的目标是优化该仪器，然后用它来研究大脑回路的组装方式。在情感、认知和创造力以及学习和记忆等重要领域的发展过程中，这种创新工具将使神经科学家能够设计实验，从而产生关于大脑线路的微妙变化如何导致毁灭性神经精神疾病的新想法。精神分裂症、自闭症、智力低下或双相情感障碍。