Trans-Sheet Illumination Microscopy (TranSIM) for decoding whole brain activity at submillisecond temporal resolution

跨片照明显微镜 (TranSIM) 以亚毫秒时间分辨率解码全脑活动

• 批准号: 9552856
• 负责人: KATSUSHI ARISAKA
• 金额: $ 14.65万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9552856
• 关键词: Accelerometer; Acoustics; Action Potentials; Algorithms; Animal Model; Area; Behavior; Behavioral; Biological Neural Networks; Brain; Caenorhabditis elegans; Complex; Computer software; Data; Detection; Development; Devices; Disease; Drosophila genus; Electron Microscopy; Electrophysiology (science); Ensure; Four-dimensional; Geometry; Health; Hour; Human; Image; Japan; Knowledge; Larva; Length; Light; Lighting; Lightning; Longevity; Maps; Methodology; Methods; Microscope; Microscopy; Monitor; Motion; Names; Nature; Neurosciences; Optics; Organism; Phototoxicity; Positioning Attribute; Research; Resolution; Sampling; Scanning; Scheme; Sepharose; Slice; Speed; Stimulus; Synapses; System; Testing; Thick; Thinness; Time; Visual; Width; Zebrafish; base; brain volume; design; experimental study; flexibility; fluorescence microscope; in vivo; information processing; innovation; interest; lens; light scattering; millisecond; next generation; optogenetics; public health relevance; relating to nervous system; sensor; temporal measurement; virtual reality; voltage

Today’s knowledge of large-scale neural networks is advancing along two orthogonal directions. Spatial (static, structural) connectomic understanding is achieved through optical and electron microscopy; yielding high spatial resolution with limited or no temporal information. Conversely, electrophysiological methods provide an exceptional temporal understanding of millisecond-order neurodynamic activities in vivo, with restrictions placed on spatial information. Unfortunately, these approaches have historically been rather mutually exclusive and incompatible with each other. This proposal is precisely aimed at breaking the methodological barrier between spatial and temporal observation, through innovative 3D optical scanning concepts which rival the temporal resolution of electrophysiology. The resulting system is ideally suited to imaging genetically expressed voltage- sensitive fluorescent markers. The proposed optical microscope is named TranSIM: Trans-Sheet Illumination Microscope. It is designed to observe brain-wide neurodynamics in model organisms (up to 1 mm3) with ~1 μm resolution in space, and sub- millisecond resolution in time. Unlike a conventional sheet illumination microscope, which illuminates a single x-y focal plane of the detection objective (on the z-axis), the conceived design forms a light sheet in the transverse direction along the z-axis (y-z plane). This sheet of light is then rapidly scanned along the x-axis as it is imaged. Multiple thin z-slices are then collected simultaneously by spatially multiplexing next-generation large-format sCMOS sensors. Regions of Interest, each with slight depth off-set from the focal plane, will map to a segmented part of the sensor, creating an eight-image focal volume in the time of a single frame. As a result, a thick brain volume can be covered by scanning the focal (x-y) plane (~1 mm2) in one direction (in x) at the frame rate of the sCMOS. The fastest sCMOS cameras are currently being jointly developed and optimized for this purpose. A line confocal readout will be implemented electrically by a rolling shutter mode of the sCMOS sensor to minimize contamination of scattering light. With proper Regions of Interest, a large volume (800 x 80 x 1000 μm3) can be scanned at 780 volumes / second; 100 times faster than today’s fastest 3D optical microscopy systems. A smaller volume (100 x 80 x 1000 μm3) can be scanned at an unprecedented rate of 6240 volumes / second, reaching the sub-millisecond temporal resolution of electrophysiological sampling. The illuminative sheet can be formed through the same detective objective for enhanced geometrical flexibility, or by an orthogonal objective (as in conventional horizontal sheet illumination) to minimize phototoxicity. In the case of a multi-view based on four-objective geometry, a large open volume (40 x 20 x 8 mm3) exists between lenses, to observe model organisms (like Zebrafish) under visual and optogenetic stimulation. Such a volume also provides smaller model organisms (C. elegans and Drosophila larvae) with ample space to navigate freely in 3D, while whole brain neural activity is monitored and controlled under various external and internal (optogenetic) stimulations.

当今对大规模神经网络的了解正在沿两个正交的方向前进。通过光学显微镜和电子显微镜实现空间（静态，结构）连接组的理解；产生高空间分辨率，没有临时信息有限或没有临时信息。相反，电生理方法提供了对体内毫秒的神经动力学活动的特殊暂时理解，并在空间信息上施加了限制。不幸的是，这些方法在历史上是相互排斥的，彼此不相容。该提案精确地旨在通过创新的3D光学扫描概念来打破空间和临时观察之间的方法论障碍，这些概念是电生理学的临时分辨率。所得系统非常适合想象一般表达的电压敏感荧光标记。 所提出的光学显微镜命名为跨表照明显微镜。它旨在观察模型生物（长达1 mm3）中的脑部神经动力学，空间分辨率约为1μm，并且时间下毫秒分辨率。与传统的板照明显微镜不同，该显微镜照亮了检测目标的单个X-y焦平面（在z轴上），构想的设计沿着Z轴（Y-Z平面）在横向方向上形成光片。然后，在成像时沿X轴迅速扫描了这张光片。然后通过空间多路复用下一代大型SCMOS传感器同时收集多个薄Z线。感兴趣的区域，每个区域都从焦平面上略有深度偏置，将映射到传感器的分段部分，在单个框架的时间内创建八模拟的焦点体积。结果，通过SCMOS的帧速率以一个方向（x）扫描焦点（X-Y）平面（〜1 mm2）可以覆盖厚的大脑体积。 目前，最快的SCMO摄像机正在共同开发和优化。线共聚焦读数将通过SCMOS传感器的滚动快门模式进行电气实现，以最大程度地减少散射光的污染。有了适当的关注区域，可以以780卷 /秒的范围扫描大量的大容量（800 x 80 x1000μm3）。比当今最快的3D光学显微镜系统快100倍。较小的体积 （100 x 80 x1000μm3）可以以6240卷 /秒的前所未有的速率扫描，以达到电生理采样的亚毫秒临时分辨率。 可以通过相同的侦探物镜形成照明纸，以增强几何柔韧性，也可以通过正交物镜（如常规水平板照明）形成，以最大程度地减少光毒性。在基于四目标几何形状的多视图的情况下，镜头之间存在较大的开放体积（40 x 20 x 8 mm3），以观察视觉和光遗传刺激下模型生物（如斑马鱼）。这样的体积还提供了较小的模型生物（秀丽隐杆线虫和果蝇幼虫），并提供足够的空间，可以在3D中自由导航，而在各种外部和内部（光学遗传）刺激下，整个脑神经元活动受到监测和控制。