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).
This project will dramatically increase the accessibility to cryo-EM by enabling high-resolution cryo-EM at 100 kV. The innovation of this project is a novel large-format (16.7 megapixel) ultra-fast electron counting direct detection camera optimized for 100 kV. This will not only enable expand the use of cryo-EM by significantly lowering the initial and ongoing cost of cryo-EM, but the additional contrast delivered by lowing the accelerating voltage will also enable high-resolution cryo-EM studies of small (<100 kDa) biomolecules, which is critical for human health research and drug discovery.