Kilohertz frame rate two-photon and confocal fluorescence microscope enabled by r

描述(申请人提供)：神经元动作电位固有的毫秒时间尺度，心脏组织中的钙波，以及蛋白质的构象变化需要具有亚毫秒时间分辨率的成像仪器来进行研究。能够在大视野内分辨最快的生物过程将使我能够研究新的方向，并更好地理解许多电生化和生化过程。多亏了荧光探针，这些事件中的许多都可以用光学显微镜观察到。然而，其微弱的荧光发射要求成像设备使用较长的积分时间来收集足够的光子来生成高信噪比的图像，这导致图像采集速度较慢。电子倍增器电荷耦合器件(EMCCD)等技术提供电子增益，以补偿在较短的积分时间内检测到的少量光子，但串行像素读出策略最终将全帧(512x512像素)速率限制在100赫兹以下。光电倍增管(PMT)提供高增益和高速读出，但通常以单元素探测器格式制造。由于这些原因，荧光显微镜一直无法以全场、衍射受限的空间分辨率来分辨毫秒级的瞬变。这项提议的目标是开发用于生物学中高速成像应用的荧光显微镜仪器。共聚焦荧光显微镜的引入，能够以足够的灵敏度和分辨率来分辨活细胞中毫秒尺度的动力学 组织将使生物学的所有领域都有新的发现。为了实现这一目标，我们建议使用射频(RF)通信领域的技术来复用样品的荧光激发和发射，以便可以使用单个PMT同时对多个像素进行成像。我们认为这项技术是使用射频多路激发的荧光成像技术，或称FIRE。虽然这项技术应该会在生物学的所有领域得到应用，但我们预计这个系统将产生最大的影响，它使新的科学成为可能，并有助于深入了解大脑和心脏的运作，其中基于荧光的钙和电压成像速度是非常重要的(例如，动作电位=1ms)。在第一年，我们将进一步开发我们的原型，以提高BOT的时间和空间分辨率。我们使用单光子激发成像固定贴壁细胞的实验的初步数据证明了这项技术的可行性，我们将把这些实验扩展到活细胞钙成像。在第二年，我们将把高速单光子成像的概念扩展到双光子激发荧光成像，使用神经元网络活动的钙成像作为原理证明。在第三年，我们将通过展示它在以前所未有的时间分辨率对乌拉坦麻醉小鼠大脑中的神经元活动进行成像的效用来展示Fire的进步。

DESCRIPTION (provided by applicant): The millisecond timescales inherent to action potentials in neurons, calcium waves in cardiac tissue, and conformational changes in proteins demand imaging instrumentation with sub-millisecond time resolution for their study. The ability to resolve the fastest biological processes over a large field of view will enable new directions i research and a better understanding of many electro-biochemical and biochemical processes. Thanks to fluorescent probes, many of these events are observable using optical microscopy. However, their weak fluorescent emission requires that imaging devices use long integration times to collect enough photons to generate images with high SNR, which result in low image acquisition speed. Technologies such as the electron-multiplier charge coupled device (EMCCD) offer electronic gain to compensate for the small number of photons detected during a shorter integration time, but the serial pixel readout strategy ultimately limits the full-frame (512x512 pixels) rate to less than 100 Hz. Photomultiplier tubes (PMTs) offer high gain and high- speed readout, but are typically manufactured in single element detector formats. For these reasons, fluorescence microscopy has been unable to resolve millisecond transients with full field, diffraction-limited spatial resolution. The goal of this proposal is to develop fluorescence microscopy instrumentation for high-speed imaging applications in biology. The introduction of a confocal fluorescence microscope capable of kilohertz frame rates with sufficient sensitivity and resolution to resolve the millisecond-timescale dynamics in living cells and tissues will enable new discoveries in all areas of biology. To accomplish this goal, we propose to employ techniques from the field of radiofrequency (RF) communications to multiplex the fluorescence excitation and emission of samples such that many pixels can be imaged simultaneously using a single PMT. We have deemed this technology Fluorescence Imaging using Radiofrequency-multiplexed Excitation, or FIRE. While this technology should find application in all areas of biology, we envision this system to make the most impact by enabling new science and aiding the development of insight into the operation of the brain and heart, where fluorescence-based calcium and voltage imaging speed are at a premium (e.g, action potential = 1 ms).In the first year, we will further develop our prototype, in order to improve bot the temporal and spatial resolutions. Our preliminary data from experiments imaging fixed adherent cells using one-photon excitation demonstrates the feasibility of this technique, and we will extend these experiments to live cell calcium imaging. In the second year, we will extend the high-speed one-photon imaging concept to two-photon excitation fluorescence imaging, using calcium imaging of neuronal network activity as proof-of-principle. During the third year, we will demonstrate the FIRE's advances by demonstrating its utility in imaging neuronal activity in the brain of urethane-anesthetized mice with unprecedented time resolution.