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

基本信息

批准号：
9091593
负责人：
Bahram Jalali
金额：
$ 18.45万
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：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9091593
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 Goals Health Heart Image Imaging Device Imaging technology In Vitro Lasers Lateral Life Measures Microscope Microscopy Mus Neurons Neurosciences Noise Optics Output Performance Photons Proteins Research Resolution Sampling 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 radio frequency radiofrequency research study sapphire laser temporal measurement two-photon voltage

项目摘要

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）：神经元动作电位、心脏组织中的钙波以及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来进行研究。在大视野范围内解析最快的生物过程的能力将为我的研究带来新的方向，并更好地理解许多电生化和生化过程。借助荧光探针，许多这些事件都可以使用光学显微镜观察到。然而，它们的荧光发射较弱，要求成像设备使用较长的积分时间来收集足够的光子以生成高信噪比的图像，从而导致图像采集速度较低。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）速率限制为更低超过 100 赫兹。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形式制造。由于这些原因，荧光显微镜无法以全场、衍射限制的空间分辨率解析毫秒瞬态。该提案的目标是开发用于生物学高速成像应用的荧光显微镜仪器。推出具有千赫兹帧速率的共焦荧光显微镜，具有足够的灵敏度和分辨率来解析活细胞中的毫秒时间尺度动态和组织将使生物学所有领域的新发现成为可能。为了实现这一目标，我们建议采用射频 (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 同时对许多像素进行成像。我们认为这项技术是使用射频复用激发（FIRE）的荧光成像技术。虽然这项技术应该在生物学的所有领域得到应用，但我们预计该系统将通过支持新科学并帮助深入了解大脑和心脏的运作而产生最大的影响，其中基于荧光的钙和电压成像速度溢价（例如，动作电位 = 1 毫秒）。在第一年，我们将进一步开发我们的原型，以提高机器人的时间和空间分辨率。我们使用单光子激发对固定贴壁细胞进行成像的实验的初步数据证明了该技术的可行性，并且我们将这些实验扩展到活细胞钙成像。第二年，我们将利用神经网络活动的钙成像作为原理验证，将高速单光子成像概念扩展到双光子激发荧光成像。在第三年，我们将通过展示 FIRE 在以前所未有的时间分辨率对聚氨酯麻醉小鼠大脑中的神经元活动进行成像方面的实用性来展示 FIRE 的进步。