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

描述（由申请人提供）：神经元动作电位固有的毫秒时间尺度，心脏组织中的钙波和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进行研究。在大视野范围内解决最快的生物过程的能力将为研究提供新的方向，并更好地理解许多电生化和生化过程。多亏了荧光探针，许多这样的事件可以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较长的集成时间来收集足够的光子以生成高信噪比的图像，这导致图像采集速度较低。电子倍增电荷耦合器件（EMCCD）等技术提供电子增益，以补偿在较短的集成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100hz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态与全视野，衍射有限的空间分辨率。本提案的目标是开发荧光显微镜仪器的高速成像应用于生物学。介绍了一种共聚焦荧光显微镜能够千赫兹帧率具有足够的灵敏度和分辨率，以解决毫秒时间尺度的动态活细胞

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.