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

r 支持的千赫兹帧速率双光子和共焦荧光显微镜

• 批准号: 8758721
• 负责人: Bahram Jalali
• 金额: $ 18.44万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8758721
• 关键词: Action Potentials; Algorithmic Software; Area; Benchmarking; Biochemical; Biochemical Process; Biological; Biological Process; Biology; Brain; Calcium; Calcium Oscillations; Cardiac; Cells; Charge; Comb animal structure; Communication; Coupled; Custom; Data; Development; Devices; Electronics; Electrons; Elements; Event; Fire - disasters; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Frequencies; Goals; Heart; Image; Imaging Device; Imaging technology; In Vitro; Lasers; Lateral; Life; Measures; Microscope; Microscopy; Mus; Neurons; Neurosciences; Noise; Optics; Output; Performance; Photons; Proteins; Radio; Research; Resolution; Sampling; Sapphire; Scanning; Science; Speed; System; Techniques; Technology; Time; Tissues; Titania; Titanium; Tube; Urethane; Validation; Work; awake; base; calcium indicator; cellular imaging; design and construction; detector; digital; fluorescence imaging; fluorescence microscope; improved; in vivo; insight; instrumentation; millisecond; operation; patch clamp; photomultiplier; photonics; prototype; public health relevance; radiofrequency; research study; two-photon; voltage

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.

描述（由申请人提供）：神经元中动作电位、心脏组织中钙波和蛋白质构象变化固有的毫秒时间尺度需要具有亚毫秒时间分辨率的成像仪器进行研究。在大视场范围内分辨最快生物过程的能力将使研究的新方向成为可能，并使人们更好地理解许多电生物化学和生物化学过程。由于有了荧光探针，许多这些事件都可以使用光学显微镜观察到。然而，它们微弱的荧光发射要求成像设备使用长的积分时间来收集足够的光子以生成具有高SNR的图像，这导致低的图像采集速度。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 Hz以下。光电倍增管（PMT）提供高增益和高速读出，但通常以单元件检测器格式制造。由于这些原因，荧光显微镜一直无法解决毫秒瞬变与全场，衍射有限的空间分辨率。本提案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。介绍了一种能够以千赫兹帧速率工作的共聚焦荧光显微镜，该显微镜具有足够的灵敏度和分辨率，可以解析活细胞中的毫秒时间尺度动力学 和组织将使生物学的各个领域都有新的发现。为了实现这一目标，我们建议采用来自射频（RF）通信领域的技术来复用样品的荧光激发和发射，使得许多像素可以使用单个PMT同时成像。我们认为这种技术是使用射频多路复用激发的荧光成像，或称为FIRE。虽然这项技术应该在生物学的所有领域都有应用，但我们设想该系统通过启用新科学并帮助开发对大脑和心脏运作的洞察力来发挥最大的影响，其中基于荧光的钙和电压成像速度非常重要。在第一年，我们将进一步开发我们的原型，以提高时间和空间分辨率。我们的初步数据从实验成像固定的粘附细胞使用单光子激发证明了这种技术的可行性，我们将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，我们将展示FIRE的进步，展示其在成像神经元活动在大脑中的麻醉小鼠前所未有的时间分辨率的实用程序。