Effect of self and extrinsic encapsulation on electron resilience of porous 2D polymer nanosheets.

Despite the exceptional resolution in aberration-corrected high-resolution transmission electron microscope (AC-HRTEM) images of inorganic two-dimensional (2D) materials, achieving high-resolution imaging of organic 2D materials remains a daunting challenge due to their low electron resilience. Optimizing the critical dose (the electron exposure, the material can accept before it is noticeably damaged) is vital to mitigate this challenge. An understanding of electron resilience in porous crystalline 2D polymers including the effect of sample thickness has not been derived thus far. It is assumed, that additional layers of the sample form a cage around inner layers, which are preventing fragments from escaping into the vacuum and enabling recombination. In the literature this so called caging effect has been reported for perylene and pythalocyanine. In this work we determine the critical dose of a porous, triazine-based 2D polymer as function of the sample thickness. The results show that the caging effect should not be generalized to more sophisticated polymer systems. We argue that pore channels in the framework structure serve as escape routes for free fragments preventing the caging effect and thus showing surprisingly a thickness-independent critical dose. Moreover, we demonstrate that graphene encapsulation prevents fragment escape and results in an increase in the critical electron dose and unit-cell image resolution.

Characterizing the resolution and throughput of the Apollo direct electron detector.

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 detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency.

One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.

Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy.

Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 µm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ∼7 Å resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the electron microscopy data bank (http://www.emdatabank.org/).

Radiation damage effects at four specimen temperatures from 4 to 100 K.

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100K. For each temperature, "exposure series" were collected consisting of consecutive images of the same area of sample, each with 10 e(-)/A(2) exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0-173.5A). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.