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

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”和具体的挑战主题06-AG-101*神经科学蓝图：开发非侵入性成像方法或技术，直接评估神经活动。它还适用于特定的挑战主题06-NS-101(开发神经活动的微创措施)和06-NS-103(神经科学的突破技术)。神经科学在21世纪面临的最大挑战之一是了解组成大脑的数十亿个神经元如何相互沟通，以产生复杂的行为。这类研究的最终好处将来自于破译神经元活动模式失调如何导致各种神经精神障碍的毁灭性症状。不幸的是，关于大脑中的神经计算如何解释感觉输入或产生与行为相关的反应，人们知之甚少。这在一定程度上是因为目前缺乏工具来审问完整大脑中大量神经元的活动。通过物理学家和神经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双光子显微镜，它结合了快速共振扫描镜和多光束成像，实现了比传统双光子显微镜快2个数量级以上的图像采集速度。为了避免深层组织双光子显微镜中多光束散射模糊的根本局限性，我们提出了一种新的方法来检测和解决不同时间不同光束的散射荧光发射。具体地说，我们将激光分成四束，然后将每束光束在光学上相互延迟3 ns。我们称这种方法为时空激发-发射复用(STEM)。所有四束光束的信号都由最先进的GHz带宽光电探测器探测到。因此，我们的显微镜保留了双光子显微镜的独特优势，包括它能够激发组织中更深层次的荧光团，它的光损伤减少，以及它精致的空间分辨率。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)能力和四色成像。最终目标是在单细胞水平上实现前所未有的6-D(x，y，z，t，？“)生物成像。此外，我们还提出了一系列活体钙实验，以系统地剖析新皮质回路的微尺度连接。首先，我们将校准我们的STEM系统，以展示其与传统的双光子钙成像相比具有更好的动作电位检测能力。接下来，我们将通过以前所未有的速度同时在2-D和3-D记录数百个这样的神经元，来研究桶形皮质中大型第2层和第3层神经元集合对胡须偏转的反应的时空动力学。在两年内，该仪器将得到优化，我们将首次能够表征新皮质体积内整个神经元的功能配置图。 与公众健康相关：我们最近开发了一种高速显微镜，可以非侵入性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这一仪器，然后用它来研究大脑回路在发育过程中是如何在对情绪、认知和创造力以及学习和记忆重要的区域组装的。这一创新工具将允许神经科学家设计实验，这些实验可以产生新的想法，即大脑连接中的细微变化如何导致破坏性的神经精神障碍，如精神分裂症、自闭症、精神发育迟滞或双相情感障碍。

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.