An Ultrafast Electron Counting Camera for 100 kV Cryo-EM

用于 100 kV 冷冻电镜的超快电子计数相机

• 批准号: 10335281
• 负责人: Benjamin Eugene Bammes
• 金额: $ 63.93万
• 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: 2021-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10335281
• 关键词: 3-Dimensional; Address; Aldehyde-Lyases; Apoferritin; Biological; COVID-19 pandemic; Capital; Collaborations; Computer software; Cryoelectron Microscopy; Data; Detection; Development; Electronics; Electrons; Emergency Situation; Equipment; Evaluation; Fast Electron; Future; Goals; Health; Human; Image; Investments; Measures; Mechanics; Microscope; Mus; Muscle; Noise; Oryctolagus cuniculus; Performance; Peripheral; Phase; Research; Research Institute; Research Personnel; Resolution; Running; Scanning Electron Microscopy; Side; Specimen; Speed; System; Techniques; Time; Validation; base; cost; cost effective; data acquisition; design; detector; drug discovery; experimental study; innovation; instrument; novel; operation; particle; printed circuit board; prototype; quantum; reconstruction; sensor; software development; structural biology; success; temporal measurement; tool; voltage

Project Summary / Abstract Single-particle electron cryo-microscopy (cryo-EM) has become an essential tool for high-resolution structural studies, both for basic biological and human health research as well as for drug discovery. Currently, one of the most pressing challenges for this powerful technique is satisfying the high and ever-growing demand for cryo-EM. This is especially challenging given the extremely high capital investment and running costs for 300 kV cryo-EM equipment. With demand for cryo-EM far exceeding capacity, there is a growing push for “democratization” of cryo-EM by developing cost-effective and highly-accessible equipment based on 100 kV TEM columns. Although the feasibility of high-resolution cryo-EM at 100 kV has been recently demonstrated (Naydenova, et al., 2019), this idea is currently hindered by the lack of suitable high-performance detectors at this low energy. Current direct detection cameras are optimized for operation for 200 and 300 kV. The performance of these cameras at 100 kV is remarkably poor, with very low resolution and very high noise due to backscattering. To address this problem, we propose to develop a new ultra-fast electron counting direct detection camera optimized for 100 kV. The proposed detector will be based on Direct Electron’s novel ultra-fast binary-readout sensor, which is capable of electron counting at an internal frame rate of up to 8,000 fps (>5× faster than any other direct detector on the market). We propose to modify the design of this detector for 100 kV operation. We have already developed an initial prototype of a 100-kV optimized direct detector based on Direct Electron’s new sensor for scanning electron microscopy (SEM), which is sensitive to 3 – 30 kV electrons. The first results from this prototype sensor confirmed that our new design delivers high resolution, minimal backscattering, and an exceptional electron counting DQE at 100 kV. During Phase II of this project, we will modify the design and layout of our novel ultra-fast binary-readout sensor to optimize it for 100 kV operation. A camera system with this new sensor will be developed for integration on common 100 kV microscopes, with the goal of minimizing both the camera’s manufacturing and on-going support costs. The new camera will be integrated in SerialEM for automated data acquisition. A workflow for high- throughput 100 kV single-particle cryo-EM will be developed. Finally, single-particle cryo-EM at 100 kV will be demonstrated using the new camera. The success of this project will create an entirely new market for high-resolution 100 kV cryo-EM. This will significantly increase the accessibility to cryo-EM, propelling structural biology forward as more researchers have access to the tools they need. Additionally, expanding the availability of cryo-EM will enable researchers to more quickly respond to future human health emergencies, such as the current COVID-19 pandemic. Finally, the additional contrast afforded by 100 kV electrons is expected to push the limits of cryo-EM of small specimens (<100 kDa).

项目概要/摘要 单粒子电子冷冻显微镜（cryo-EM）已成为高分辨率结构研究的重要工具 研究，既用于基础生物学和人类健康研究，也用于药物发现，目前是其中之一。 这种强大技术最紧迫的挑战是满足对冷冻电镜不断增长的高需求。 鉴于 300 kV 冷冻电镜的资本投资和运行成本极高，这一点尤其具有挑战性 由于对冷冻电镜的需求远远超过了产能，人们越来越多地推动冷冻电镜的“民主化”。 通过开发基于 100 kV TEM 柱的经济高效且易于使用的设备来实现冷冻电镜。 尽管最近已经证明了 100 kV 高分辨率冷冻电镜的可行性（Naydenova 等人， 2019），这一想法目前因缺乏合适的低能量电流高性能探测器而受到阻碍。 直接检测相机针对 200 kV 和 300 kV 的操作进行了优化。 100 kV 的效果非常差，分辨率非常低，并且由于反向散射而产生的噪声非常高。 为了解决这个问题，我们建议开发一种新型超快电子计数直接检测相机 所提出的探测器将针对 100 kV 进行优化，基于 Direct Electron 的新型超快速二进制读出技术。 传感器，能够以高达 8,000 fps 的内部帧速率进行电子计数（比任何传感器快 5 倍） 我们建议修改该探测器的设计以实现 100 kV 操作。 我们已经开发了基于 Direct Electron 的 100 kV 优化直接探测器的初始原型 用于扫描电子显微镜 (SEM) 的新型传感器，对 3 – 30 kV 电子敏感。 该原型传感器证实我们的新设计可提供高分辨率、最小的反向散射和 100 kV 下出色的电子计数 DQE。 在该项目的第二阶段，我们将修改新型超快速二进制读出传感器的设计和布局 为了优化其 100 kV 操作，将开发带有这种新传感器的相机系统以集成到 常见的 100 kV 显微镜，目标是最大限度地减少相机的制造和持续支持 新相机将集成到 SerialEM 中以实现高自动化数据采集工作流程。 将开发吞吐量为 100 kV 的单粒子冷冻电镜，最终将开发出 100 kV 的单粒子冷冻电镜。 使用新相机进行演示。 该项目的成功将为高分辨率 100 kV 冷冻电镜创造一个全新的市场。 显着提高冷冻电镜的可及性，随着越来越多的研究人员的研究，推动结构生物学向前发展 此外，扩大冷冻电镜的可用性将使研究人员能够获得更多。 快速应对未来的人类卫生紧急情况，例如当前的 COVID-19 大流行。 100 kV 电子提供的额外对比度有望突破小样本冷冻电镜的极限 （<100 kDa）。