Open research questions in Advanced Electron Microscopy Techniques and Applications

What the literature leaves open

• The role of interparticle proximity in sintering remains insufficiently resolved. Prior work has focused on strategies targeting intraparticle morphology or particle-support interactions, but has rarely incorporated interparticle proximity. The general view is that postmortem observations provide only end-state structures, making it difficult to elucidate the restructuring mechanism.

  Pseudotime uncovers restructuring dynamics and mitigators of metal catalyst deactivation · 2026 · DOI

• Electron-event representation (EER) data can be rendered at half the physical pixel size, but whether super-resolution processing can recover useful information beyond the physical Nyquist limit from noisy in situ data remains unclear.

  Super-Resolution Processing Enables In Situ Subtomogram Averaging Beyond the Nyquist Limit · 2026 · DOI

• Although substrate-engaged structures have revealed key steps in OMP assembly, how substrate-free BamA closes its lateral gate and which conformations it adopts in a lipid bilayer remain unclear.

  CLOSING THE CONFORMATIONAL CYCLE OF THE BETA-BARREL ASSEMBLY MACHINERY · 2026 · DOI

• Substitution of hydrogen by deuterium halves this recoil energy and makes the bonds involved slightly harder to break, yet its protective effect on vitrified protein crystals has not been tested.

  Replacing H2O with D2O in cryo-EM reduces radiation damage in biological samples · 2026 · DOI

• The direct application of state-space models to anisotropic EM data has been limited. Existing methods have insufficient global modeling capabilities or local feature extraction capabilities. There is a need for a novel network that adapts state-space models to anisotropic 3D EM images.

  MambaSSM: efficient segmentation of brain structures in anisotropic 3D EM images via state-space models · 2026 · DOI

• Traditional Cryo-EM reconstruction methods are based on maximum likelihood approaches, which can be time-consuming and limited in resolution. Deep learning-based methods have begun to emerge, but there is still a need for improved methods.

  CryoFIFA: deep curriculum learning for cryo-EM fine and fast ab-initio reconstruction · 2026 · DOI

• The existence of the quantum offset in velocity imaging-based electron spectrometry is unclear. The accuracy of velocity imaging-based electron spectrometry measurements is uncertain.

  Evaluation of quantum offset in velocity imaging-based electron spectrometry · 2026 · DOI

• The need to optimize the signal-to-noise ratio in low-dose conditions.

  Enhancing Dose Efficiency of Optimum Bright‐Field Scanning Transmission Electron Microscopy Using a Phase‐Shifted Electron Probe · 2026 · DOI

• The need for a review of the benefits and limitations of cryo-EM in battery research.

  Freeze it or leave it? Evaluating the role of cryo-electron microscopy in battery research · 2026 · DOI

• Future research will focus on expanding the high-quality and diverse cryo-EM annotated dataset to improve the model’s generalization capability. Future work will explore integrating a classifier into SAM to increase particle picking accuracy. However, the model’s performance is limited by the scale and diversity of the training data.

  CryoPromptSeg: Prompt-guided Segmentation with Integrated Denoising for Cryo-EM Particle Picking · 2026 · DOI

• The lack of a direct way to assign chemical identity to densities observed in electron micrographs. The need for a correlative workflow combining cryo-EM and MS for integrated spatiochemical analysis of biological samples.

  Subcellular chemical mapping using correlated cryogenic electron and mass spectrometry imaging · 2026 · DOI

• The lack of methods that can enhance local detail and connectivity in cryo-EM maps without suppressing weak but biologically relevant structural context. The need for a statistically grounded measure of reliability for cryo-EM maps.

  Confidence-guided cryo-EM map optimisation with LocScale-2.0 · 2026 · DOI

• Existing super-resolution methods require extensive calibration and retraining for each imaging setup. The need for a device-agnostic deep-learning framework that eliminates the need for calibration data or explicit knowledge of optical system parameters.

  From stars to molecules: AI guided device-agnostic super-resolution imaging · 2026 · DOI

• The risk of structural hallucination remains a critical concern for data-driven methods. The risk of overfitting to ideal secondary structures remains a concern.

  EMReady2: improvement of cryo-EM and cryo-ET maps by local quality-aware deep learning with Mamba · 2026 · DOI

• Identifying comprehensive thermodynamic behaviors of biomacromolecules remains challenging. Large structural changes occur on millisecond or longer timescales in general, making MD simulation often unrealistic. The dynamic behaviors of biomacromolecules span a wide range of timescale and magnitudes, making quantitative analysis difficult.

  Digital twin of biomacromolecular thermodynamics in cryo-EM data · 2026 · DOI

• To develop new techniques that can analyze mechanical properties at the nano scale.

  Electron Backscattered Diffraction Analysis and Image Correlation Analysis using Scanning Electron Microscopy for Understanding the Mechanical Properties in Materials · 2026 · DOI

• Several opportunities exist to further enhance AFMap-UNet performance, generalization, and usability. Architectural optimization and incorporation of more efficient backbones, such as EfficientNet or MobileNet, could reduce computational costs without compromising accuracy, facilitating implementation in laboratories with limited GPU resources.

  AFMap-UNet enables accurate nuclear segmentation of atomic force microscopy images with minimal training data · 2026 · DOI

• Improving cryoFIFA for small particles using image denoising and tight masks. Exploring the potential of cryoFIFA for future applications in Cryo-EM analysis. Developing new deep learning-based methods for Cryo-EM reconstruction.

  CryoFIFA: deep curriculum learning for cryo-EM fine and fast ab-initio reconstruction · 2026 · DOI

• Further development of the BPC method to improve its accuracy and robustness. Application of the BPC method to other types of electron detectors. Investigation of the BPC method's potential in improving crystallography metrics and structure solutions.

  Robust electron counting for direct electron detectors with the Back-propagation counting method · 2026 · DOI

• The method may not perform well with very noisy or low-resolution cryo-EM density maps, - The method requires a significant amount of computational resources and expertise, - The method is limited to medium-resolution cryo-EM density maps, - Deep-learning methods may require larger labelled datasets and extensive…

  A Novel Machine-Learning Based Method for Resolving Secondary Structure Topology in Medium-Resolution Cryo-EM Density Maps · 2026 · DOI

• Exploring the application of deep-learning methods for map segmentation, SSE annotation, and backbone tracing, - Investigating the use of other classification algorithms and feature spaces, - Developing methods to improve the accuracy of SSE correspondence in very noisy or low-resolution cryo-EM density…

  A Novel Machine-Learning Based Method for Resolving Secondary Structure Topology in Medium-Resolution Cryo-EM Density Maps · 2026 · DOI

• The use of the cryo-EM-FIB-SIMS workflow for studying the uptake and storage of other chemical pollutants by environmental bacteria. The exploration of the use of the workflow for subcellular localization of molecules in eukaryotic cells.

  Subcellular chemical mapping using correlated cryogenic electron and mass spectrometry imaging · 2026 · DOI

• Applying the method to other types of scientific measurements. Investigating the potential of the method in improving image clarity and reducing measurement time. Exploring the application of the method in other fields, such as medical diagnostics.

  Deep prior-based denoising for state-of-the-art scientific imaging and metrology · 2026 · DOI

• Combining spIsoNet with experimental strategies to alleviate the severe preferred-orientation problem - Applying spIsoNet to other cryoEM datasets with limited angular…

  Using spIsoNet to address the preferred-orientation problem in cryoEM reconstructions · 2026 · DOI

• Further development of LocScale-2.0 and LocScale-FEM to improve their accuracy and robustness. Application of the method to a wider range of cryo-EM maps and specimens.

  Confidence-guided cryo-EM map optimisation with LocScale-2.0 · 2026 · DOI

Most-cited papers in Advanced Electron Microscopy Techniques and Applications

• Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction · Nature Methods · 2020 · 2,011 citations
• DeepEMhancer: a deep learning solution for cryo-EM volume post-processing · Communications Biology · 2021 · 1,470 citations
• New tools for automated cryo-EM single-particle analysis in RELION-4.0 · Biochemical Journal · 2021 · 1,266 citations
• 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM · Journal of Structural Biology · 2021 · 1,088 citations
• Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION -3.1 · IUCrJ · 2020 · 948 citations
• Single-particle cryo-EM at atomic resolution · Nature · 2020 · 910 citations
• Atomic-resolution protein structure determination by cryo-EM · Nature · 2020 · 793 citations
• CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks · Nature Methods · 2021 · 689 citations
• Topaz-Denoise: general deep denoising models for cryoEM and cryoET · Nature Communications · 2020 · 635 citations
• Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells · Nature Methods · 2021 · 576 citations

Most recent work

• NeuVue: A scalable and customizable framework for electron microscopy proofreading · bioRxiv · 2026
• Information in 4D-STEM: Where it is, and How to Use it · Ultramicroscopy · 2026
• AlphaFold as a prior: experimental structure determination conditioned on a pretrained neural network · Nature Methods · 2026
• Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET · Nature Protocols · 2026
• Cryo-EM: the revolution continues · IUCrJ · 2026
• Noise2Void for denoising atomic resolution scanning transmission electron microscopy images · npj Computational Materials · 2026
• Compartment‐guided assembly of large‐scale molecular models with Bentopy · Protein Science · 2026
• Round robin on structure analysis from 3D electron diffraction data · IUCrJ · 2026
• Deconvolution improves cryo-EM maps · bioRxiv · 2026
• De novo identification of protein domains in cryo-electron tomography maps from AlphaFold2 models · bioRxiv · 2026

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