Electron cryomicroscopy (cryo-EM) has revolutionized structural biology, enabling near-atomic resolution 3D structures of biological macromolecules. Conventional cryo-EM uses conventional transmission electron microscopy (CTEM) with large underfocus to enhance contrast, followed by computationally intensive image processing to combine numerous projections into a high-resolution 3D structure. However, scanning transmission electron microscopy (STEM), particularly integrated differential phase contrast STEM (IDPC-STEM), offers potential advantages. STEM techniques have demonstrated exceptional resolution in materials science, reaching sub-angstrom levels. IDPC-STEM, in particular, is known for its high contrast, direct image interpretability (without defocusing), and dose efficiency, even for beam-sensitive materials. While STEM has been applied to some biological samples, full 3D single-particle cryo-EM structures at near-atomic resolution hadn't been achieved until this study. This research investigated whether IDPC-STEM could be a valuable alternative to CTEM for high-resolution cryo-EM, addressing the need for efficient and high-resolution imaging methods in structural biology. The purpose is to explore a novel approach for single-particle cryo-EM, potentially improving resolution, speed, and efficiency. The importance lies in the potential to expand the capabilities of cryo-EM, particularly for challenging samples or where rapid analysis is critical.

The authors review existing literature on STEM and cryo-EM. They highlight previous work demonstrating high-resolution capabilities of various STEM techniques, including ptychography, for dose-resistant samples. Several studies successfully applied IDPC-STEM to various materials, including beam-sensitive ones like zeolites and metal-organic frameworks, achieving high-resolution imaging with low electron doses. Previous STEM applications to biological samples, however, were limited in resolution or scope. Some studies used STEM tomography on thick vitrified cells, while others examined single-particle specimens or viruses with ptychography, but at low resolution. The authors acknowledge the success of CTEM in cryo-EM, particularly its advancements in hardware and software resulting in the 'resolution revolution' allowing for routine near-atomic resolution. They cite examples of high-resolution CTEM structures of test specimens like TMV and apoferritin. This review sets the stage for the current study by highlighting both the strengths of established cryo-EM and the potential benefits of a novel STEM-based approach.

The study used two biological specimens, KLH and TMV, for cryo-EM structure determination using IDPC-STEM. The methodology involved several key steps: 1. **Specimen Preparation:** Quantifoil grids were prepared using a glow discharge device to make them hydrophilic, facilitating even specimen distribution. KLH and TMV solutions were applied and blotted using a Vitrobot Mark IV before plunge freezing in liquid nitrogen. 2. **STEM Imaging:** Imaging was conducted on a Thermo Fisher Scientific Titan Krios G4 operating at 300 kV. The column was equipped with a high-brightness field emission gun, a three-condenser lens system, and a Panther segmented STEM detector. Different convergence semi-angles (CSAs) were used to control the resolution, with gold standards used to calibrate resolution. Beam alignment and center-of-mass (COM) determination were critical steps. The total electron dose was carefully controlled (35 e⁻/Ų) to minimize radiation damage. 3. **IDPC-STEM Acquisition:** The four-quadrant mode of the Panther STEM detector was employed to obtain IDPC-STEM images. Precise alignment of the detector and scan direction were crucial. The acquisition software (Velox v.3.2) and MAPS software allowed for efficient image acquisition and correlation of TEM and STEM data. The study employed a simple line-by-line scan grid. 4. **Image Processing and 3D Reconstruction:** Raw IDPC-STEM micrographs were preprocessed using a high-pass Gaussian filter. For KLH, single-particle analysis was performed using CryoSPARC v.3.3.1, involving particle picking, 2D and 3D classification, and refinement. For TMV, helical coordinates were picked in EMAN2, power spectra were analyzed to calibrate pixel size, and Relion v.3.1 was used for helical reconstruction. CTF correction was not needed for IDPC-STEM data due to the continuously transferred contrast. 5. **Validation:** The obtained structures were compared with previously determined CTEM structures of TMV from EMPIAR-10305 and EMPIAR-10021 data sets, validating the accuracy and resolution. Simulations were also performed to evaluate factors affecting resolution and signal-to-noise ratio, considering variables such as CSA, pixel size, and electron dose. The authors meticulously describe experimental parameters, including CSAs, pixel sizes, electron doses, acquisition times, and software used at each stage.

The key findings demonstrate the feasibility and effectiveness of using IDPC-STEM for high-resolution cryo-EM structure determination. Specifically: * **High-resolution structures:** Near-atomic resolution structures were successfully obtained for both KLH (6.5 Å) and TMV (3.5 Å) using IDPC-STEM. The TMV resolution was comparable or better than some previous CTEM datasets. * **Continuous contrast transfer:** IDPC-STEM images showed continuous contrast transfer over the entire frequency range, unlike CTEM, which has oscillating CTF. This simplifies image processing and eliminates the need for CTF correction. * **Improved low-resolution contrast:** For smaller CSAs, IDPC-STEM demonstrated enhanced low-resolution contrast, potentially beneficial for more complex samples. * **Resolution dependence on CSA:** The study clearly showed that increasing the CSA increased resolution, following theoretical predictions. However, higher CSAs decrease depth of focus, potentially creating depth sections rather than full projections, and introduce other aberrations. * **Comparable radiation damage:** The radiation damage effects observed in the iDPC-STEM maps were comparable to those seen in CTEM maps, indicating that the method does not substantially increase damage. * **Superior map quality:** The 4.0 mrad iDPC-STEM TMV map at 3.5Å exhibited similar quality to a 2019 CTEM acquisition and better quality than the 2015 CTEM acquisition (EMPIAR-10021). * **Efficient imaging (potential):** While initial acquisition times were longer than standard CTEM, the authors note that faster detectors and automation could significantly reduce acquisition times, potentially making it comparable to CTEM. These results demonstrate that IDPC-STEM offers a promising alternative to CTEM for high-resolution cryo-EM and highlights its potential for various applications. The detailed quantitative analysis and comparison with CTEM data provide strong support for these findings.

The successful determination of near-atomic resolution structures of KLH and TMV using IDPC-STEM strongly supports its viability as a high-resolution cryo-EM technique. The consistent contrast transfer across all frequencies, avoiding the oscillating CTF of CTEM, simplifies image processing and potentially reduces computational demands. The study's comparison with established CTEM datasets demonstrates comparable or even superior performance in some cases, particularly concerning map quality. The demonstrated resolution is within theoretical limits based on CSA and other factors, validating both the approach and the methodology. The dependence of resolution on CSA is in line with theoretical predictions. While initially slower acquisition times are a limitation, potential improvements through automation and faster detectors are promising. This work opens exciting possibilities for cryo-EM, especially for samples where maximizing low-resolution contrast or mitigating beam-induced motion are crucial. Future exploration into application of IDPC-STEM for thicker samples and tomographic reconstruction are promising directions.

This research successfully demonstrates the use of IDPC-STEM for high-resolution single-particle cryo-EM structure determination. Near-atomic resolution structures of KLH and TMV were obtained, comparable to or exceeding the resolution of some established CTEM methods. The study highlights the benefits of continuous contrast transfer, simplified image processing, and potential for improved low-resolution contrast. Future studies should focus on automation, faster detectors, application to thicker samples, and tomographic reconstructions. This technique holds immense potential for advancing cryo-EM and its applications in structural biology.

The current study uses a limited number of samples. While the results are promising, further studies with diverse biological macromolecules are needed to fully evaluate the generalizability of the technique. Although the potential for faster acquisition speeds exists with advancements in detectors and automation, the current acquisition times are slightly longer than traditional CTEM methods. The study primarily focuses on single-particle analysis, and future studies should explore its applicability for more complex samples or situations where averaging may be more challenging, such as cellular cryo-electron tomography. The resolution achieved, while impressive, still lags behind the very highest resolutions obtained by cutting-edge CTEM. Further optimization of experimental parameters and detector technology might be needed to reach those levels consistently.