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：直接评估神经活动的非侵入性成像方法或技术的发展。它也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（神经科学的突破性技术）。21世纪神经科学面临的最大挑战之一是理解构成大脑的数十亿神经元如何相互沟通，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之间功能失调的活动模式如何导致各种神经精神疾病的破坏性症状。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行为相关反应，我们知之甚少。这部分是由于目前缺乏工具来询问完整大脑中大量神经元的活动。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率。为了避免深层组织双光子显微镜中多光束散射模糊的基本限制，我们提出了一种创新的方法来检测和分辨不同时间不同光束的散射荧光发射。具体来说，我们将激光束分成四束，然后将每束与其他光束的光学延迟3ns。我们称这种方法为时空激发发射多路复用（STEM）。来自所有四束的信号由最先进的GHz带宽光电探测器检测。因此，我们的显微镜保留了双光子显微镜的独特优势，包括能够激发组织中更深的荧光团，减少光损伤和精致的空间分辨率。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，我们提出了一系列体内钙实验来系统地剖析新皮层回路的微观连接。首先，我们将校准我们的STEM系统，以证明与传统的双光子钙成像相比，其优越的动作电位检测。接下来，我们将通过以前所未有的速度同时记录数百个这些神经元的二维和三维数据，研究桶状皮层中第2层和第3层神经元大集合对晶须挠曲的时空动态反应。在2年内，该仪器将得到优化，我们将能够首次表征一个体积的新皮层内整个神经元补体的功能接线图。

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.