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: 10325452
• 负责人: Benjamin Eugene Bammes
• 金额: $ 110.49万
• 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 2023-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325452
• 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.

项目总结/摘要 了解大脑功能和神经系统疾病的前提是映射之间的连接， 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。这种研究跨越了广泛的尺度，既需要一个大的领域- of-view用于绘制连通性，高分辨率用于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良好的应用前景 分辨率，同时还检测感兴趣的特定分子组分。该技术类似于多- 彩色荧光显微镜，但在约100×更高的放大倍数。然而，目前的方法 获取多色EM数据是基于能量过滤TEM（EFTEM）的，这显著地限制了其在以下方面的有用性： 由于其极低的通量和有限的视野， 我们建议开发一种新的超快速直接检测相机扫描电子显微镜（SEM）的能力 以每秒超过100，000帧（fps）的速度运行，并测量检测到的电子的能量。等 相机将是一个惊人的飞跃，大大提高吞吐量，并使复杂的多， 彩色EM技术，使用连续块面SEM（SBEM），使小结构，如突触囊泡， 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。了初步成效 证实灵敏度电子下降到2千伏的能量，显示出远远优于上级的信息内容相比， 最先进的扫描电镜相机，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。这些初步结果被用来最后确定 本文提出的新的超快像素化直接检测器的要求，其速度需要 使该技术可用于大视场、高分辨率多色SBEM以成像神经元。 在第二阶段，我们将推进这种新型超快速SEM相机的开发和商业化。 系统，通过制造和组装新的超快SEM相机，进一步完善硬件和软件， 有效地处理产生的大量数据并识别多色EM标签，然后 展示了神经元组织的高速多色SBEM。 这个项目的成功将创造一个无处不在的荧光显微镜技术的模拟，但在 使用连续块面SEM的分辨率显著更高。这不仅将有广泛的应用， 神经科学研究，但也将扩展到细胞显微镜在广泛的其他生物领域。 此外，新相机还将实现能量过滤电子背散射衍射（EBSD）， 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， 我们预期所提出的检测器将在多个领域中具有广泛的影响。