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We describe the development, operation, and application of the 4D Camera-a 576 by 576 pixel active pixel sensor for scanning/transmission electron microscopy which operates at 87,000 Hz. The detector generates data at ∼480 Gbit/s which is captured by dedicated receiver computers with a parallelized software infrastructure that has been implemented to process the resulting 10-700 Gigabyte-sized raw datasets. The back illuminated detector provides the ability to detect single electron events at accelerating voltages from 30 to 300 kV. Through electron counting, the resulting sparse data sets are reduced in size by 10--300× compared to the raw data, and open-source sparsity-based processing algorithms offer rapid data analysis. The high frame rate allows for large and complex scanning diffraction experiments to be accomplished with typical scanning transmission electron microscopy scanning parameters.

Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.

• direct electron detectors produce clusters of pixel hits for each single electron. • a flow-counting algorithm with adaptive buffer size enable real-time analysis. • electron arrival times in continuous beams follow Poisson statistics. • femtosecond multi-electron pulses have anti-correlation effects. Event-driven hybrid pixel detectors with nanosecond time resolution have opened up novel pathways in modern ultrafast electron microscopy, for example in hyperspectral electron-energy loss spectroscopy or free-electron quantum optics. However, the impinging electrons typically excite more than one pixel of the device, and an efficient algorithm is therefore needed to convert the measured pixel hits to real single-electron events. Here we present a robust clustering algorithm that is fast enough to find clusters in a continuous stream of raw data in real time. Each tuple of position and arrival time from the detector is continuously compared to a buffer of previous hits until the probability of a merger with an old event becomes irrelevant. In this way, the computation time becomes independent of the density of electron arrival and the algorithm does not break the operation chain. We showcase the performance of the algorithm with a 'timepix' camera in two regimes of electron microscopy, in continuous beam emission and laser-triggered femtosecond mode. [ABSTRACT FROM AUTHOR]

2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10–12 Å can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending , refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5 Å. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware. These developments also greatly increased the usability and applicability of CET as a diagnostic and research tool, which is now enabling structural biologists to determine the structure of proteins in their native cellular environment to sub-nanometer resolution. These recent technical developments have stimulated us to update on our previous review (Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine. Ann Anat 191, 427–445) in which we described the fundamentals of CET. In this follow-up, we extend this basic description in order to explain the aforementioned recent advances, and describe related 3D techniques that can be applied to the anatomy of biological systems that are relevant for medicine. [ABSTRACT FROM AUTHOR]

Since the development of parallel electron energy loss spectroscopy (EELS), chargecoupled devices (CCDs) have been the default detectors for EELS. With the recent development of electron-counting direct-detection cameras, micrographs can be acquired under very low electron doses at significantly improved signal-to-noise ratio. In spectroscopy, in particular in combination with a monochromator, the signal can be extremely weak and the detection limit is principally defined by noise introduced by the detector. Here we report the use of an electron-counting direct-detection camera for EEL spectroscopy. We studied the oxygen K edge of amorphous ice and obtained a signal noise ratio up to 10 times higher than with a conventional CCD. We report the application of electron counting to record time-resolved EEL spectra of a biological protein embedded in amorphous ice, revealing chemical changes observed in situ while exposed by the electron beam. A change in the fine structure of nitrogen K and the carbon K edges were recorded during irradiation. A concentration of 3 at% nitrogen was detected with a total electron dose of only 1.7 e-/Ų, extending the boundaries of EELS signal detection at low electron doses. [ABSTRACT FROM AUTHOR]

Hybrid pixel direct electron detectors are gaining popularity in electron microscopy due to their excellent properties. Some commercial cameras based on this technology are relatively affordable which makes them attractive tools for experimentation especially in combination with an SEM setup. To support this, a detector characterization (Modulation Transfer Function, Detective Quantum Efficiency) of an Advacam Minipix and Advacam Advapix detector in the 15–30 keV range was made. In the current work we present images of Point Spread Function, plots of MTF/DQE curves and values of DQE(0) for these detectors. At low beam currents, the silicon detector layer behaviour should be dominant, which could make these findings transferable to any other available detector based on either Medipix2, Timepix or Timepix3 provided the same detector layer is used. • Timepix DED is excellent at 15–30 keV, PSF-MTF are near ideal, max DQE(0) is 60% • PSF-MTF degrade with e-beam energy increase, PSF-MTF improve with DED threshold rise • DQE values show a significant DQE reduction at increased detector threshold value • At 15keV beam energy both cameras show low DQE due to Al layer on top of sensor [ABSTRACT FROM AUTHOR]

Ultrafast-optical-pump — structural-probe measurements, including ultrafast electron and x-ray scattering, provide direct experimental access to the fundamental timescales of atomic motion, and are thus foundational techniques for studying matter out of equilibrium. High-performance detectors are needed in scattering experiments to obtain maximum scientific value from every probe particle. We deploy a hybrid pixel array direct electron detector to perform ultrafast electron diffraction experiments on a WSe 2 /MoSe 2 2D heterobilayer, resolving the weak features of diffuse scattering and moiré superlattice structure without saturating the zero order peak. Enabled by the detector's high frame rate, we show that a chopping technique provides diffraction difference images with signal-to-noise at the shot noise limit. Finally, we demonstrate that a fast detector frame rate coupled with a high repetition rate probe can provide continuous time resolution from femtoseconds to seconds, enabling us to perform a scanning ultrafast electron diffraction experiment that maps thermal transport in WSe 2 /MoSe 2 and resolves distinct diffusion mechanisms in space and time. • We perform ultrafast electron diffraction on a WSe 2 /MoSe 2 heterobilayer • We denoise diffraction data with high frame rate direct electron detection • Diffraction data resolves a 10 nm periodicity moiré pattern • High dynamic range allows simultaneous measurement of atomic-scale Bragg peaks • Scanning micro-diffraction shows dynamics from angstrom to millimeter length scales [ABSTRACT FROM AUTHOR]

Conventional techniques for structural analysis have intrinsic limitations, including damage or distortion of the functional conformation of a protein complex during purification steps. Recently, a promising electron microscopy technique, cryo-electron tomography (cryo-ET), has been used to obtain insights regarding the molecular assemblies that drive cellular processes. This review focuses on the introduction of the cryo-ET and its recent improvement of resolution, in combination with the development of hardware and software such as direct electron detectors and sub-tomogram averaging techniques. Although cryo-ET still has several challenges, it can provide a glimpse into the interaction and function of macromolecules in their native environment. [ABSTRACT FROM AUTHOR]

The use of a direct electron detector for the simple acquisition of 2D electron backscatter diffraction (EBSD) maps and 3D EBSD datasets with a static sample geometry has been demonstrated in a focused ion beam scanning electron microscope. The small size and flexible connection of the Medipix direct electron detector enabled the mounting of sample and detector on the same stage at the short working distance required for the FIB. Comparison of 3D EBSD datasets acquired by this means and with conventional phosphor based EBSD detectors requiring sample movement showed that the former method with a static sample gave improved slice registration. However, for this sample detector configuration, significant heating by the detector caused sample drift. This drift and ion beam reheating both necessitated the use of fiducial marks to maintain stability during data acquisition. [ABSTRACT FROM AUTHOR]

Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%. The use of direct electron detectors is growing very rapidly: they were used for 70% of the structures released in 2016, in contrast to none before 2011. Microscopes from FEI have an overwhelming lead in terms of usage, and the use of the RELION software package continues to grow rapidly after having attained a leading position in the field. China is rapidly emerging as a major player in the field, supplementing the US, Germany and the UK as the big four. Similarly, Tsinghua University ranks only second to the MRC Laboratory for Molecular Biology in terms of involvement in publications associated with cryo-EM structures at better than 4 Å resolution. Overall, the numbers point to a rapid democratization of the field, with more countries and institutes becoming involved. [ABSTRACT FROM AUTHOR]

• We evaluate the performance of a monolithic active pixel sensor direct electron detector (DED) for the characterization of short-range order (SRO) in nanoscale volumes of amorphous materials, using SiO 2 and Ta 2 O 5 thin films as test cases. • The performance of the DED is compared systematically to electron scattering measurements recorded on an indirect detector (CCD) using 200 and 80 keV electrons. • We demonstrate that the performance of the DED resulted in improved SRO characterization in comparison to that obtained from the CCD measurements, namely a larger measured maximal scattering vector, and improved signal-to-noise ratio across all spatial frequencies. These improvements are shown to result from the superior detective quantum efficiency of the DED. Consequently, the DED measurements enabled to determine the coordination numbers of atomic bonds more accurately. • In addition, the effects of sample cooling and energy-filtering on the measured SRO of the oxides were investigated. The introduction of direct electron detectors (DEDs) to transmission electron microscopy has set off the ' resolution revolution ', especially for cryoTEM low-dose imaging of soft matter. In comparison to traditional indirect electron detectors such as Charged-Coupled Devices (CCD), DEDs show an improved modulation transfer function (MTF) and detective quantum efficiency (DQE) across all spatial frequencies, as well as faster frame rates which enable single electron counting. The benefits of such characteristics for imaging, spectroscopy and electron holography have been demonstrated previously. However, studies are lacking on the application of DEDs for localized characterization of short- to medium- range-order (SRO, MRO) in amorphous materials using electron scattering. Therefore, we evaluate the performance of a Monolithic Active Pixel Sensor DED for the characterization of SRO and MRO in nanoscale volumes of amorphous materials, using SiO 2 and Ta 2 O 5 thin films as test cases. The performance of the detector is compared systematically to electron scattering measurements recorded on an indirect detector (CCD) using 200 keV electrons and electron doses starting at approximately 500 e − Å 2 . In addition, the effects of sample cooling and energy-filtering on the measured SRO of the oxides were investigated. We demonstrate that the performance of the DED resulted in improved SRO characterization in comparison to that obtained from the CCD measurements. The DED enabled to achieve a larger measured maximal scattering vector, ∼ 16.5 1 Å compared to ∼ 15 1 Å , for the CCD. Furthermore, an improved signal-to-noise ratio of approximately two-fold was observed across all spatial frequencies for both 200 keV and 80 keV electrons. These improvements are shown to result from the superior DQE of the DED. Consequently, the DED measurements enabled to determine the coordination numbers of atomic bonds more accurately. We expect that further benefits of the DED for S/MRO characterization will be highlighted for ultra- sensitive materials that cannot withstand electron doses above several e − Å 2 . [ABSTRACT FROM AUTHOR]

[Display omitted] • Gatan K2/K3 direct electron detectors used for MicroED in counting mode. • Macromolecular structures determined to atomic resolution. • No beam stop was used on K3 and no apparent damage to the camera observed. Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED. [ABSTRACT FROM AUTHOR]

Summary CorA, the major Mg 2+ uptake system in prokaryotes, is gated by intracellular Mg 2+ (K D ∼1–2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg 2+ -bound and Mg 2+ -free conditions, but EPR spectroscopic studies reveal large Mg 2+ -driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg 2+ -bound closed conformation and in two open Mg 2+ -free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg 2+ , four of the five subunits are displaced to variable extents (∼10–25 Å) by hinge-like motions as large as ∼35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg 2+ , open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels. [ABSTRACT FROM AUTHOR]

As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8 Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model. [ABSTRACT FROM AUTHOR]

In early 2011 FEI Company launched the “Falcon”, its first commercial direct electron detector product intended for application in 3-D electron microscopy in the life sciences. In this paper we discuss the principle of direct electron detection and its implementation in Falcon cameras. We describe the signal formation in the sensor and its impact on the detection quantum efficiency (DQE) of the sensor. Insights into the signal formation led us to improved camera designs. Three significant improvements are discussed. (1) Back thinning of the sensor. This is implemented in the second-generation Falcon (Falcon 2), where the sensor thickness is reduced to 50 μm, and in the latest generation Falcon 3 detector with further back-thinning down to 30 μm. (2) The introduction of electron counting, a signal processing technology implemented in Falcon 3. (3) Dose fractionation mode, which allows the user to access intermediate results during the illumination of the sample. [ABSTRACT FROM AUTHOR]

Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets. [ABSTRACT FROM AUTHOR]

Direct detector device (DDD) cameras have revolutionized single particle electron cryomicroscopy (cryo-EM). In addition to an improved camera detective quantum efficiency, acquisition of DDD movies allows for correction of movement of the specimen, due to both instabilities in the microscope specimen stage and electron beam-induced movement. Unlike specimen stage drift, beam-induced movement is not always homogeneous within an image. Local correlation in the trajectories of nearby particles suggests that beam-induced motion is due to deformation of the ice layer. Algorithms have already been described that can correct movement for large regions of frames and for >1 MDa protein particles. Another algorithm allows individual <1 MDa protein particle trajectories to be estimated, but requires rolling averages to be calculated from frames and fits linear trajectories for particles. Here we describe an algorithm that allows for individual <1 MDa particle images to be aligned without frame averaging or linear trajectories. The algorithm maximizes the overall correlation of the shifted frames with the sum of the shifted frames. The optimum in this single objective function is found efficiently by making use of analytically calculated derivatives of the function. To smooth estimates of particle trajectories, rapid changes in particle positions between frames are penalized in the objective function and weighted averaging of nearby trajectories ensures local correlation in trajectories. This individual particle motion correction, in combination with weighting of Fourier components to account for increasing radiation damage in later frames, can be used to improve 3-D maps from single particle cryo-EM. [ABSTRACT FROM AUTHOR]

Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for cryo-electron tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed cryo-electron tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in cryo-electron tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells. [ABSTRACT FROM AUTHOR]

Improved electron detectors and image-processing techniques will allow the structures of macromolecules to be determined from tens of thousands of single-particle cryo-EM images, rather than the hundreds of thousands needed previously.

Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.

Abstract: The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction. [Copyright &y& Elsevier]

• Assessing cryo-electron micrographs using the spectral signal-to-noise ratio. • Measuring the effect of residual movement after movie frame alignment. • Quantifying radiation damage using the progressive spectral signal-to-noise ratio. The quality of a cryoEM reconstruction is fundamentally a function of the signal-to-noise ratio (SNR) of the original micrographs. The SNR embodies multiple aspects of image formation, including microscope details and alignment, specimen composition and thickness, how it is recorded, and how the specimen degrades during imaging. With the advent of direct electron detectors and the recording of a series of images for each micrograph (a movie), we have an opportunity to count every electron and derive fully quantitative results. After alignment of the movie frames of a micrograph, we can calculate the SNR, or its spatial frequency equivalent, the spectral SNR (SSNR). This SSNR reflects residual movement between frames and the progressive effect of radiation damage. The goal is to develop a quantitative analysis of the SSNR and radiation damage to assess and improve the quality of micrographs. Several test cases were selected from the EMPIAR database and ten micrograph movies downloaded for each case. The movie frames were aligned as rigid bodies to compensate for stage and support movement. The SSNR for subsets of frames was then calculated to assess the effect of residual movement. The progressive SSNR (PSSNR) was subsequently calculated to determine the decrease in signal accumulation as a result of radiation damage. In all cases the alignment of the movie frames compensated for global movement to the extent that the effect on the SSNR is negligible. The subset SSNR can be used as a tool to further confirm the extent of residual movement. The progressive SSNR indicates an increase in value up to an asymptote, consistent with the theory for radiation damage. Fitting these curves gives the inherent SNR before exposure, and the critical dose, which decreases with spatial frequency with an exponential parameter roughly between one and two. The implementation of the PSSNR for movie frames provides a tool for assessing micrograph quality and progression of radiation damage. The estimation of the critical dose further quantifies radiation damage and may shed some light on the mechanisms of damage. These are likely both a function of the specimen composition and the imaging parameters used. [ABSTRACT FROM AUTHOR]

Abstract: A new generation of direct electron detectors for transmission electron microscopy (TEM) promises significant improvement over previous detectors in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE). However, the performance of these new detectors needs to be carefully monitored in order to optimize imaging conditions and check for degradation over time. We have developed an easy-to-use software tool, FindDQE, to measure MTF and DQE of electron detectors using images of a microscope’s built-in beam stop. Using this software, we have determined the DQE curves of four direct electron detectors currently available: the Gatan K2 Summit, the FEI Falcon I and II, and the Direct Electron DE-12, under a variety of total dose and dose rate conditions. We have additionally measured the curves for the Gatan US4000 and TVIPS TemCam-F416 scintillator-based cameras. We compare the results from our new method with published curves. [Copyright &y& Elsevier]

TEAM I is the final product of the Transmission Electron Aberration-corrected Microscope (TEAM) Project, a collaborative project funded by the Department of Energy with the goal of designing and building a platform for a next generation aberration-corrected electron microscope capable of image resolution of up to 50 pm. The TEAM instrument incorporates a number of new technologies, including spherical- and chromatic-aberration correction, an all-piezo-electric sample stage and an active-pixel direct electron detector. This article describes the functionality of this advanced instrumentation, its response to changes in environment or operating conditions, and its stability during daily operation within the National Center for Electron Microscopy user facility. [ABSTRACT FROM AUTHOR]

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease., (© 2021 Wiley-VCH GmbH.)

Abstract: Step improvements in electron detectors are needed for transmission electron microscopes (TEM) to take full advantage of latest developments in electron optics and electron sources. This work presents beam tests performed with column parallel charge-coupled devices (CPCCD) and discusses measured cluster properties. Excellent signal-to-noise ratio is found, demonstrating that CPCCD are well suited for TEM. Large variations in cluster-integrated intensity and size are observed, attributed to the strong scattering of 120–200keV electrons in silicon. Spatial momentum analysis of clusters reveals strong asymmetry indicating that position resolution could be limited for certain cluster shapes. [Copyright &y& Elsevier]

• Hybrid pixel direct detector is near-ideal for electron energy loss spectroscopy. • Large dynamic range, fast frame rate, zero dark current and readout noise. • Record both inner shell loss edges and intense zero loss peak without saturation. • Fast elemental mapping with no noise penalty for summing many frames. • Efficient parallel acquisition with high DQE for angle resolved EELS. We characterize a hybrid pixel direct detector and demonstrate its suitability for electron energy loss spectroscopy (EELS). The detector has a large dynamic range, narrow point spread function, detective quantum efficiency ≥ 0.8 even without single electron arrival discrimination, and it is resilient to radiation damage. It is capable of detecting ~5 × 106 electrons/pixel/second, allowing it to accommodate up to 0.8 pA per pixel and hence >100 pA EELS zero-loss peak (ZLP) without saturation, if the ZLP is spread over >125 pixels (in the non-dispersion direction). At the same time, it can reliably detect isolated single electrons in the high loss region of the spectrum. The detector uses a selectable threshold to exclude low energy events, and this results in essentially zero dark current and readout noise. Its maximum frame readout rate at 16-bit digitization is 2250 full frames per second, allowing for fast spectrum imaging. We show applications including EELS of boron nitride in which an unsaturated zero loss peak is recorded at the same time as inner shell loss edges, elemental mapping of an STO/BTO/LMSO multilayer, and efficient parallel acquisition of angle-resolved EEL spectra (S(q, ω)) of boron nitride. [ABSTRACT FROM AUTHOR]

• The performance of direct electron detector (K2 IS) has been compared with the charge coupled device (CCD) detector at both 200 kV and 80 kV. • The advantages of using K2 detectors become more obvious when operating at higher accelerating voltage. • The spectroscopic application of direct electron detector will help the microscopy field extract bonding and electronic structures of difficult materials (beam sensitive/ charging/ easy contaminated/ fast drifting samples) at higher resolution. Direct electron detectors (DeDs) have been widely used for imaging studies because of their higher beam sensitivity, lower noise, improved pixel resolution, etc. However, there have been limited studies related to the performance in spectroscopic applications for the direct electron detection. Hereby, taking the advantage of the DeD installed on a high-performance electron energy-loss spectrometer, we systematically studied the performance of a DeD (Gatan's K2 IS) fitted on an aberration-corrected transmission electron microscope (TEM) equipped with an electron monochromator. Using SrTiO 3 as the model system, the point spread function in the zero-loss region of the spectrum and the performance for core loss spectroscopy have been investigated under both 200 kV and 80 kV operating conditions. We demonstrate that the K2 detector can achieve an overall better performance at 200 kV than a charge coupled device (CCD) detector. At 80 kV, the K2 DeD is still better than a CCD, except for the relative broad tails of the zero-loss peak. The signal-to-noise ratio is very close for DeD and CCD under 80 kV. Based on our data obtained at different operating voltages, it is clear that DeD will benefit the microscopy community and boost the development of cutting-edge materials science studies by pushing the frontiers in electron energy-loss spectroscopy. [ABSTRACT FROM AUTHOR]

• The Medipix3 is a hybrid counting fast (≥ 12k frames per second) pixelated detector. • Noise-free readout enables recording of single electron impacts at short exposures. • Detector physics & configuration relevant to time resolution are discussed. • Demonstration of sub-100ns time resolution in stroboscopic imaging. • Applicable in standard (S)TEMs, maintaining optimum imaging conditions. Detector developments are currently enabling new capabilities in the field of transmission electron microscopy (TEM). We have investigated the limits of a hybrid pixel detector, Medipix3, to record dynamic, time varying, electron signals. Operating with an energy of 60 keV, we have utilised electrostatic deflection to oscillate electron beam position on the detector. Adopting a pump-probe imaging strategy, we have demonstrated that temporal resolutions three orders of magnitude smaller than are available for typically used TEM imaging detectors are possible. Our experiments have shown that energy deposition of the primary electrons in the hybrid pixel detector limits the overall temporal resolution. Through adjustment of user specifiable thresholds or the use of charge summing mode, we have obtained images composed from summing 10,000s frames containing single electron events to achieve temporal resolution less than 100 ns. We propose that this capability can be directly applied to studying repeatable material dynamic processes but also to implement low-dose imaging schemes in scanning transmission electron microscopy. [ABSTRACT FROM AUTHOR]

Many nanoparticles in fields such as heterogeneous catalysis undergo surface structural fluctuations during chemical reactions, which may control functionality. These dynamic structural changes may be ideally investigated with time-resolved in situ electron microscopy. We have explored approaches for extracting quantitative information from large time-resolved image data sets with a low signal to noise recorded with a direct electron detector on an aberration-corrected transmission electron microscope. We focus on quantitatively characterizing beam-induced dynamic structural rearrangements taking place on the surface of CeO2 (ceria). A 2D Gaussian fitting procedure is employed to determine the position and occupancy of each atomic column in the nanoparticle with a temporal resolution of 2.5 ms and a spatial precision of 0.25 Å. Local rapid lattice expansions/contractions and atomic migration were revealed to occur on the (100) surface, whereas (111) surfaces were relatively stable throughout the experiment. The application of this methodology to other materials will provide new insights into the behavior of nanoparticle surface reconstructions that were previously inaccessible using other methods, which will have important consequences for the understanding of dynamic structure-property relationships.

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware. These developments also greatly increased the usability and applicability of CET as a diagnostic and research tool, which is now enabling structural biologists to determine the structure of proteins in their native cellular environment to sub-nanometer resolution. These recent technical developments have stimulated us to update on our previous review (Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine. Ann Anat 191, 427-445) in which we described the fundamentals of CET. In this follow-up, we extend this basic description in order to explain the aforementioned recent advances, and describe related 3D techniques that can be applied to the anatomy of biological systems that are relevant for medicine., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Direct detector device (DDD) cameras dramatically enhance the capabilities of electron cryomicroscopy (cryo-EM) due to their improved detective quantum efficiency (DQE) relative to other detectors. DDDs use semiconductor technology that allows micrographs to be recorded as movies rather than integrated individual exposures. Movies from DDDs improve cryo-EM in another, more surprising, way. DDD movies revealed beam-induced specimen movement as a major source of image degradation and provide a way to partially correct the problem by aligning frames or regions of frames to account for this specimen movement. In this chapter, we use a self-consistent mathematical notation to explain, compare, and contrast several of the most popular existing algorithms for computationally correcting specimen movement in DDD movies. We conclude by discussing future developments in algorithms for processing DDD movies that would extend the capabilities of cryo-EM even further., (© 2016 Elsevier Inc. All rights reserved.)

Macromolecular electron microscopy typically depicts the structures of macromolecular complexes ranging from ∼200 kDa to hundreds of MDa. The amount of specimen required, a few micrograms, is typically 100 to 1000 times less than needed for X-ray crystallography or nuclear magnetic resonance spectroscopy. Micrographs of frozen-hydrated (cryogenic) specimens portray native structures, but the original images are noisy. Computational averaging reduces noise, and three-dimensional reconstructions are calculated by combining different views of free-standing particles ("single-particle analysis"). Electron crystallography is used to characterize two-dimensional arrays of membrane proteins and very small three-dimensional crystals. Under favorable circumstances, near-atomic resolutions are achieved. For structures at somewhat lower resolution, pseudo-atomic models are obtained by fitting high-resolution components into the density. Time-resolved experiments describe dynamic processes. Electron tomography allows reconstruction of pleiomorphic complexes and subcellular structures and modeling of macromolecules in their cellular context. Significant information is also obtained from metal-coated and dehydrated specimens., (Copyright © 2015 John Wiley & Sons, Inc.)