An energy discriminating direct detector for multi-color SEM

用于多色 SEM 的能量辨别直接探测器

• 批准号: 10474559
• 负责人: Benjamin Eugene Bammes
• 金额: $ 75.17万
• 依托单位: DIRECT ELECTRON, LP
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474559
• 关键词: Advanced Development; Algorithms; Area; Biological; Biological Markers; Brain; Cells; Characteristics; Collaborations; Color; Computer software; Coupled; Data; Detection; Development; Discrimination; Electron Microscopy; Electronics; Electrons; Energy-Filtering Transmission Electron Microscopy; Equipment; Event; Face; Fluorescence Microscopy; Generations; Glutamates; Head; Image; Industrialization; Label; Location; Maps; Measurement; Measures; Mechanics; Mental disorders; Methods; Microscopy; Molecular; Nervous System Physiology; Neurobiology; Neurodegenerative Disorders; Neurons; Neurosciences Research; Noise; Nuclear Pore; Nucleosomes; Optics; Output; Parkinson Disease; Phase; Population; Positioning Attribute; Quality Control; Research; Resolution; Scanning; Scanning Electron Microscopy; Solid; Specimen; Speed; Structure; Surface; Synapses; Synaptic Vesicles; System; Techniques; Technology; Testing; Time; Tissues; Validation; Virus; analog; base; cellular imaging; commercialization; cost; data acquisition; design; detector; electron energy; experience; experimental study; improved; innovation; interest; materials science; method development; microscopic imaging; nanometer; nanometer resolution; nervous system disorder; novel; protein protein interaction; prototype; relating to nervous system; sensor; software development; spectral energy; success

Project Summary / Abstract Understanding brain function and neurological disorder is predicated on mapping the connectivity among neurons, distinguishing various cellular and molecular populations, and elucidating the protein-protein interactions that drive neurological function. Such studies span a wide range of scales, requiring both a large field- of-view to map connectivity and high-resolution to visualize subcellular and intrasynaptic molecular details. Multi-color electron microscopy (EM) has shown promise in studying biological ultrastructure at nanometer resolution while also detecting specific molecular components of interest. The technique is analogous to multi- color fluorescence microscopy, but at about ~100× higher magnification. However, the current method for acquiring multi-color EM data is based on energy-filtered TEM (EFTEM), which significantly limits is usefulness in neurobiology due to its severely low throughput and limited field-of-view. We propose to develop a new ultra-fast direct detection camera for scanning electron microscopy (SEM) capable of operating at more than 100,000 frames per second (fps) and measuring the energy of detected electrons. Such a camera will be an astounding leap forward, dramatically improving throughput and enabling sophisticated multi- color EM techniques using serial block-face SEM (SBEM), so that small structures like synaptic vesicles, nucleosomes, nuclear pores, and viruses (all a few nanometers to 10-40 nm) can be identified and quantified. We have already developed a Phase I prototype of this new direct detection SEM camera, based on a low-energy- optimized version of Direct Electron’s current generation TEM direct detection cameras. Initial results have confirmed sensitivity to electrons down to 2 kV energy, showed far superior information content compared to current state-of-the-art scintillator-coupled SEM cameras, and most importantly, revealed that our new sensor design is capable of energy discrimination of detected electrons. These initial results were used to finalize the requirements for the new ultra-fast pixelated direct detector proposed here, the speed of which is required to make the technique useful for large field-of-view, high-resolution multi-color SBEM for imaging neurons. During Phase II we will advance the development and commercialization of this new ultra-fast SEM camera system, by fabricating and assembling the new ultra-fast SEM camera, further refining hardware and software to efficiently handle the enormous volumes of data produced and identify multi-color EM labels, and then demonstrating high-speed multi-color SBEM of neuronal tissue. The success of this project will create an analog of the ubiquitous fluorescence light microscopy technique, but at significantly higher resolution using serial block-face SEM. This will not only have wide ranging applications for neuroscience research but will also extend to cellular microscopy in a wide range of other biological fields. Additionally, the new camera will also enable energy-filtered electron backscattered diffraction (EBSD), which is widely used in materials science research and industrial quality control. Therefore, as a new enabling technology, we anticipate that the proposed detector will have broad impact across a variety of fields.

项目概要/摘要 了解大脑功能和神经系统疾病的基础是绘制之间的连接性 神经元，区分各种细胞和分子群体，并阐明蛋白质-蛋白质 驱动神经功能的相互作用。此类研究涉及范围广泛，需要大范围的研究 视野图可绘制连通性，高分辨率可可视化亚细胞和突触内分子细节。 多色电子显微镜 (EM) 在研究纳米生物超微结构方面显示出前景 分辨率，同时还可以检测感兴趣的特定分子成分。该技术类似于多 彩色荧光显微镜，但放大倍数约为 100 倍。然而，目前的方法 获取多色 EM 数据基于能量过滤 TEM (EFTEM)，这极大地限制了其用途 神经生物学由于其通量极低且视野有限。 我们建议开发一种新型超快直接检测相机，用于扫描电子显微镜（SEM） 以超过 100,000 帧每秒 (fps) 的速度运行并测量检测到的电子的能量。这样的 相机将是一次惊人的飞跃，极大地提高吞吐量并实现复杂的多 使用串行块面 SEM (SBEM) 的彩色 EM 技术，使得像突触囊泡这样的小结构， 核小体、核孔和病毒（均为几纳米至 10-40 nm）可以被识别和定量。 我们已经开发了这种新型直接检测 SEM 相机的第一阶段原型，该原型基于低能量- Direct Electron 当前一代 TEM 直接检测相机的优化版本。初步结果有 证实对低至 2 kV 能量的电子的敏感性，与 当前最先进的闪烁体耦合 SEM 相机，最重要的是，揭示了我们的新传感器 设计能够对检测到的电子进行能量区分。这些初步结果用于最终确定 这里提出了对新型超快像素化直接探测器的要求，其速度要求 使该技术可用于大视场、高分辨率多色 SBEM 神经元成像。 在第二阶段，我们将推进这种新型超快 SEM 相机的开发和商业化 系统，通过制造和组装新型超快SEM相机，进一步完善硬件和软件 有效处理产生的大量数据并识别多色 EM 标签，然后 展示神经元组织的高速多色 SBEM。 该项目的成功将创造出无处不在的荧光光学显微镜技术的模拟，但在 使用串行块面 SEM 可以显着提高分辨率。这不仅具有广泛的应用 神经科学研究，但也将扩展到广泛的其他生物领域的细胞显微镜。 此外，新相机还将实现能量过滤电子背散射衍射（EBSD），这是 广泛应用于材料科学研究和工业质量控制。因此，作为一种新的使能技术， 我们预计所提出的探测器将在各个领域产生广泛的影响。