A Novel Event-Based Active Pixel Sensor for Cryo-EM Electron Counting
Microscopy and Microanalysis
The introduction and widespread adoption of electron counting with direct detectors, along with improvements in software for image processing and 3D reconstruction, have propelled a "resolution revolution" in cryo-EM  , so that atomic models of biological macromolecules are now routinely generated by the technique  . As a result, cryo-EM has become an essential tool in both basic biological research as well as drug discovery , with demand for cryo-EM equipment often exceeding
... ty  . One of the most significant bottlenecks for cryo-EM is the restrictive imaging conditions imposed by electron counting. Currently, electron counting is performed by acquiring a large number of frames from a high-sensitivity direct detector, with the electron beam sufficiently dim to yield sparse images of single electron events on each frame   . The sparsity within each frame is necessary to avoid coincidence loss stemming from the inability to discriminate multiple coincident electrons as separate events [5, 7] . Operating outside of the sparse conditions for the camera has detrimental consequences, particularly for quantitative, low-dose applications such as cryo-EM. Therefore, at present, cryo-EM experiments are largely dictated by the specific exposure rate requirements of the electron counting camera. Rethinking the strategy for electron counting, we have developed and implemented a novel large format (4096 × 4096) event-based monolithic active pixel sensor (MAPS) direct detection sensor (Fig. 1) . Noise is minimized through on-chip correlated double sampling (CDS) and on-chip thresholding. Incident electrons are detected through an optimized sense amplifier, which encodes each detection event and sends it through high-speed digital input/output channels to on-board field programmable gate arrays (FPGAs), which perform sub-pixel precision centroiding on each event and accumulate super-resolution (8192 × 8192) dose-fractionated frames to be sent to the computer. To maximize resolution, this new sensor has a larger pixel size (8 μm) than our previous direct detection sensors so that incident TEM primary electrons have a higher probability of being constrained a small number of pixels. This novel sensor can detect and process incident electrons at an order of magnitude higher rate than brute force software-based counting detectors. The new detector maintains >95% linearity up to 60 electrons per pixel per second at 200 and 300 kV, with high detective quantum efficiency ( >90%) over at least three orders of magnitude of exposure rates (Fig. 2) . The camera has been successfully integrated in SerialEM for automated acquisition of both single particle and tomographic cryo-EM data. This new strategy for electron counting promises to significantly improve the productivity and throughput of cryo-EM, by enabling high-speed acquisition of high-SNR datasets. Additionally, the simplicity of onchip electron counting reduces the overall cost of this camera, making high-resolution cryo-EM equipment more accessible. https://www.cambridge.org/core/terms. https://doi.