An Ultrafast Electron Counting Camera for 100 kV Cryo-EM

项目摘要/摘要 单粒子电子冷冻显微镜(Cryo-EM)已成为研究高分辨率结构的重要工具 研究，包括基础生物学和人类健康研究以及药物发现。目前，其中一家 对于这项强大的技术来说，最紧迫的挑战是满足对冷冻-EM不断增长的高需求。 考虑到300千伏低温EM极高的资本投资和运行成本，这一点尤其具有挑战性 设备。随着对低温EM的需求远远超过产能，人们越来越强烈地推动低温EM的“民主化” CRYO-EM通过开发基于100千伏瞬变电磁柱的高性价比和高可访问性设备。 尽管最近已经证明了100千伏高分辨率低温电磁的可行性(Naydenova等人， 2019年)，这一想法目前因缺乏合适的高性能探测器而受到阻碍。当前 直接探测摄像机针对200千伏和300千伏的工作电压进行了优化。这些摄像头的性能在 100千伏非常差，分辨率非常低，由于后向散射，噪声非常高。 为了解决这一问题，我们提出了一种新型的超快电子计数直接探测相机 优化为100千伏。建议的探测器将基于Direct Electron的新型超快二进制读出 传感器，能够以高达8,000 fps的内部帧速率进行电子计数(&gt；比任何 市场上的其他直接检测器)。我们建议对该探测器的设计进行修改，以适应100千伏的工作。 我们已经开发了基于Direct Electron的100千伏优化直接探测器的初步样机 新型扫描电子显微镜传感器，对3-30千伏的电子很敏感。第一个结果来自 这款原型传感器证实了我们的新设计提供了高分辨率、最小的后向散射和 在100千伏时，电子计数异常。 在这个项目的第二阶段，我们将修改我们的新型超高速二进制读出传感器的设计和布局 以优化它以适应100千伏的运行。将开发一个带有这种新传感器的摄像系统，以集成到 普通的100千伏显微镜，目标是最大限度地减少摄像机的制造和持续支持 成本。新的摄像头将集成在SerialEM中，以实现自动数据采集。适用于高- 将开发生产能力为100千伏的单粒子低温电磁场。最后，100千伏的单粒子低温电磁场将 演示了如何使用新相机。 该项目的成功将为高分辨率100千伏冷冻-EM创造一个全新的市场。这将是 极大地提高了低温电磁的可及性，推动了结构生物学的发展，因为越来越多的研究人员 获得他们所需的工具。此外，扩大冷冻-EM的可用性将使研究人员能够 快速应对未来人类健康突发事件，例如当前的新冠肺炎大流行。最后， 100千伏电子提供的额外对比度有望突破小样品的低温电磁场极限 (&lt；100 kDa)。

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).