Accurate determination of three-dimensional (3D) atomic structures is crucial for understanding and controlling the properties of nanomaterials. Atomic electron tomography (AET) offers non-destructive atomic imaging with picometer-level precision, enabling the resolution of defects, interfaces, and strain fields in 3D, as well as the observation of dynamic structural evolution. However, reconstruction artifacts arising from geometric limitations and electron dose constraints can hinder reliable atomic structure determination. Recent progress has integrated deep learning, especially convolutional neural networks, into AET workflows to improve reconstruction fidelity. This review highlights recent advances in neural network-assisted AET, emphasizing its role in overcoming persistent challenges in 3D atomic imaging. By significantly enhancing the accuracy of both surface and bulk structural characterization, these methods are advancing the frontiers of nanoscience and enabling new opportunities in materials research and technology.
Cryo-electron tomography (cryo-ET) has emerged as a powerful technique for imaging macromolecular complexes in their near-native states. However, the localization of 3D particles in cellular environments still presents a significant challenge due to low signal-to-noise ratios and missing wedge artifacts. Deep learning approaches have shown great potential, but they need huge amounts of data, which can be a challenge in cryo-ET scenarios where labeled data is often scarce. In this paper, we propose a novel Self-augmented and Self-interpreted (SaSi) deep learning approach towards few-shot particle detection in 3D cryo-ET images. Our method builds upon self-augmentation techniques to further boost data utilization and introduces a self-interpreted segmentation strategy for alleviating dependency on labeled data, hence improving generalization and robustness. As demonstrated by experiments conducted on both simulated and real-world cryo-ET datasets, the SaSi approach significantly outperforms existing state-of-the-art methods for particle localization. This research increases understanding of how to detect particles with very few labels in cryo-ET and thus sets a new benchmark for few-shot learning in structural biology.
Cryo-electron tomography (cryoET) is a crucial technique for unveiling the structure of protein complexes. Automatically analyzing tomograms captured by cryoET is an essential step toward understanding cellular structures. In this paper, we introduce the 4th place solution from the CZII - CryoET Object Identification competition, which was organized to advance the development of automated tomogram analysis techniques. Our solution adopted a heatmap-based keypoint detection approach, utilizing an ensemble of two different types of 2.5D U-Net models with depth reduction. Despite its highly unified and simple architecture, our method achieved 4th place, demonstrating its effectiveness.
Cryo-electron tomography (cryo-ET) enables 3D visualization of cellular environments. Accurate reconstruction of high-resolution volumes is complicated by the very low signal-to-noise ratio and a restricted range of sample tilts, creating a missing wedge of Fourier information. Recent self-supervised deep learning approaches, which post-process initial reconstructions done by filtered backprojection (FBP), have significantly improved reconstruction quality, but they are computationally expensive, demand large memory, and require retraining for each new dataset. End-to-end supervised learning is an appealing alternative but is impeded by the lack of ground truth and the large memory demands of high-resolution volumetric data. Training on synthetic data often leads to overfitting and poor generalization to real data, and, to date, no general end-to-end deep learning reconstructors exist for cryo-ET. In this work, we introduce CryoLithe, a local, memory-efficient reconstruction network that directly estimates the volume from an aligned tilt-series, overcoming the suboptimal FBP. We demonstrate that leveraging transform-domain locality makes our network robust to distribution shifts, enabling effective supervised training and giving excellent results on real data -- without retraining or fine-tuning.
Cryo-Electron Tomography (Cryo-ET) enables detailed 3D visualization of cellular structures in near-native states but suffers from low signal-to-noise ratio due to imaging constraints. Traditional denoising methods and supervised learning approaches often struggle with complex noise patterns and the lack of paired datasets. Self-supervised methods, which utilize noisy input itself as a target, have been studied; however, existing Cryo-ET self-supervised denoising methods face significant challenges due to losing information during training and the learned incomplete noise patterns. In this paper, we propose a novel self-supervised learning model that denoises Cryo-ET volumetric images using a single noisy volume. Our method features a U-shape J-invariant blind spot network with sparse centrally masked convolutions, dilated channel attention blocks, and volume unshuffle/shuffle technique. The volume-unshuffle/shuffle technique expands receptive fields and utilizes multi-scale representations, significantly improving noise reduction and structural preservation. Experimental results demonstrate that our approach achieves superior performance compared to existing methods, advancing Cryo-ET data processing for structural biology research
The Bundle Adjustment (BA) model is commonly optimized using a nonlinear least squares method, with the Levenberg-Marquardt (L-M) algorithm being a typical choice. However, despite the L-M algorithm's effectiveness, its sensitivity to initial conditions often results in slower convergence when applied to poorly conditioned datasets, motivating the exploration of alternative optimization strategies. This paper introduces a novel algorithm for optimizing the BA model in the context of image sequence alignment for cryo-electron tomography, utilizing optimal control theory to directly optimize general nonlinear functions. The proposed Optimal Control Algorithm (OCA) exhibits superior convergence rates and effectively mitigates the oscillatory behavior frequently observed in L-M algorithm. Extensive experiments on both synthetic and real-world datasets were conducted to evaluate the algorithm's performance. The results demonstrate that the OCA achieves faster convergence compared to the L-M algorithm. Moreover, the incorporation of a bisection-based update procedure significantly enhances the OCA's performance, particularly in poorly initialized datasets. These findings indicate that the OCA can substantially improve the efficiency of 3D reconstructions in cryo-electron tomography.
Cryo-electron microscopy (cryo-EM) is a powerful technique in structural biology and drug discovery, enabling the study of biomolecules at high resolution. Significant advancements by structural biologists using cryo-EM have led to the production of over 38,626 protein density maps at various resolutions1. However, cryo-EM data processing algorithms have yet to fully benefit from our knowledge of biomolecular density maps, with only a few recent models being data-driven but limited to specific tasks. In this study, we present CryoFM, a foundation model designed as a generative model, learning the distribution of high-quality density maps and generalizing effectively to downstream tasks. Built on flow matching, CryoFM is trained to accurately capture the prior distribution of biomolecular density maps. Furthermore, we introduce a flow posterior sampling method that leverages CRYOFM as a flexible prior for several downstream tasks in cryo-EM and cryo-electron tomography (cryo-ET) without the need for fine-tuning, achieving state-of-the-art performance on most tasks and demonstrating its potential as a foundational model for broader applications in these fields.
Three-dimensional atomic resolution imaging using transmission electron microscopes is a unique capability that requires challenging experiments. Linear electron tomography methods are limited by the missing wedge effect, requiring a high tilt range. Multislice ptychography can achieve deep sub-{\AA}ngstrom resolution in the transverse direction, but the depth resolution is limited to 2 to 3 nanometers. In this paper, we propose and demonstrate an end-to-end approach to reconstructing the electrostatic potential volume of the sample directly from the 4D-STEM datasets. End-to-end multi-slice ptychographic tomography recovers several slices at each tomography tilt angle and compensates for the missing wedge effect. The algorithm is initially tested in simulation with a Pt@$\mathrm{Al_2O_3}$ core-shell nanoparticle, where both heavy and light atoms are recovered in 3D from an unaligned 4D-STEM tilt series with a restricted tilt range of 90 degrees. We also demonstrate the algorithm experimentally, recovering a Te nanoparticle with sub-{\AA}ngstrom resolution.
Cryogenic Electron Tomography (CryoET) is a useful imaging technology in structural biology that is hindered by its need for manual annotations, especially in particle picking. Recent works have endeavored to remedy this issue with few-shot learning or contrastive learning techniques. However, supervised training is still inevitable for them. We instead choose to leverage the power of existing 2D foundation models and present a novel, training-free framework, CryoSAM. In addition to prompt-based single-particle instance segmentation, our approach can automatically search for similar features, facilitating full tomogram semantic segmentation with only one prompt. CryoSAM is composed of two major parts: 1) a prompt-based 3D segmentation system that uses prompts to complete single-particle instance segmentation recursively with Cross-Plane Self-Prompting, and 2) a Hierarchical Feature Matching mechanism that efficiently matches relevant features with extracted tomogram features. They collaborate to enable the segmentation of all particles of one category with just one particle-specific prompt. Our experiments show that CryoSAM outperforms existing works by a significant margin and requires even fewer annotations in particle picking. Further visualizations demonstrate its ability when dealing with full tomogram segmentation for various subcellular structures. Our code is available at: https://github.com/xulabs/aitom
Integrative modeling enables structure determination for large macromolecular assemblies by combining data from multiple sources of experiment data with theoretical and computational predictions. Recent advancements in AI-based structure prediction and electron cryo-microscopy have sparked renewed enthusiasm for integrative modeling; structures from AI-based methods can be integrated with in situ maps to characterize large assemblies. This approach previously allowed us and others to determine the architectures of diverse macromolecular assemblies, such as nuclear pore complexes, chromatin remodelers, and cell-cell junctions. Experimental data spanning several scales was used in these studies, ranging from high-resolution data, such as X-ray crystallography and Alphafold structures, to low-resolution data, such as cryo-electron tomography maps and data from co-immunoprecipitation experiments. Two recurrent modeling challenges emerged across a range of studies. First, modeling disordered regions, which constituted a significant portion of these assemblies, necessitated the development of new methods. Second, methods needed to be developed to utilize the information from cryo-electron tomography, a timely challenge as structural biology is increasingly moving towards in situ characterization. Here, we recapitulate recent developments in the modeling of disordered proteins and the analysis of cryo-electron tomography data and highlight opportunities for method development in the context of integrative modeling.
Cryo-Electron Tomography (cryo-ET) is a 3D imaging technology facilitating the study of macromolecular structures at near-atomic resolution. Recent volumetric segmentation approaches on cryo-ET images have drawn widespread interest in biological sector. However, existing methods heavily rely on manually labeled data, which requires highly professional skills, thereby hindering the adoption of fully-supervised approaches for cryo-ET images. Some unsupervised domain adaptation (UDA) approaches have been designed to enhance the segmentation network performance using unlabeled data. However, applying these methods directly to cryo-ET images segmentation tasks remains challenging due to two main issues: 1) the source data, usually obtained through simulation, contain a certain level of noise, while the target data, directly collected from raw-data from real-world scenario, have unpredictable noise levels. 2) the source data used for training typically consists of known macromoleculars, while the target domain data are often unknown, causing the model's segmenter to be biased towards these known macromolecules, leading to a domain shift problem. To address these challenges, in this work, we introduce the first voxel-wise unsupervised domain adaptation approach, termed Vox-UDA, specifically for cryo-ET subtomogram segmentation. Vox-UDA incorporates a noise generation module to simulate target-like noises in the source dataset for cross-noise level adaptation. Additionally, we propose a denoised pseudo-labeling strategy based on improved Bilateral Filter to alleviate the domain shift problem. Experimental results on both simulated and real cryo-ET subtomogram datasets demonstrate the superiority of our proposed approach compared to state-of-the-art UDA methods.
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of the huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim, and long mitochondria observed in healthy conditions transforms into isolated, swollen, and short entities, with internal cristae disorganization, cavities, and abnormally large matrix granules.
Understanding the structures of biological macromolecules is highly important as they are closely associated with cellular functionalities. Comprehending the precise organization actin filaments is crucial because they form the dynamic cytoskeleton, which offers structural support to cells and connects the cell's interior with its surroundings. However, determining the precise organization of actin filaments is challenging due to the poor quality of cryo-electron tomography (cryo-ET) images, which suffer from low signal-to-noise (SNR) ratios and the presence of missing wedge, as well as diverse shape characteristics of actin filaments. To address these formidable challenges, the primary component of this dissertation focuses on developing sophisticated computational techniques for tracing actin filaments. In particular, three novel methodologies have been developed: i) BundleTrac, for tracing bundle-like actin filaments found in Stereocilium, ii) Spaghetti Tracer, for tracing filaments that move individually with loosely cohesive movements, and iii) Struwwel Tracer, for tracing randomly orientated actin filaments in the actin network. The second component of the dissertation introduces a convolutional neural network (CNN) based segmentation model to determine the location of protein secondary structures, such as helices and beta-sheets, in medium-resolution (5-10 Angstrom) 3-dimensional cryo-electron microscopy (cryo-EM) images. This methodology later evolved into a tool named DeepSSETracer. The final component of the dissertation presents a novel algorithm, cylindrical fit measure, to estimate image structure match at helix regions in medium-resolution cryo-EM images. Overall, my dissertation has made significant contributions to addressing critical research challenges in structural biology by introducing various computational methods and tools.
We introduce ICE-TIDE, a method for cryogenic electron tomography (cryo-ET) that simultaneously aligns observations and reconstructs a high-resolution volume. The alignment of tilt series in cryo-ET is a major problem limiting the resolution of reconstructions. ICE-TIDE relies on an efficient coordinate-based implicit neural representation of the volume which enables it to directly parameterize deformations and align the projections. Furthermore, the implicit network acts as an effective regularizer, allowing for high-quality reconstruction at low signal-to-noise ratios as well as partially restoring the missing wedge information. We compare the performance of ICE-TIDE to existing approaches on realistic simulated volumes where the significant gains in resolution and accuracy of recovering deformations can be precisely evaluated. Finally, we demonstrate ICE-TIDE's ability to perform on experimental data sets.
Cryogenic electron tomography is a technique for imaging biological samples in 3D. A microscope collects a series of 2D projections of the sample, and the goal is to reconstruct the 3D density of the sample called the tomogram. Reconstruction is difficult as the 2D projections are noisy and can not be recorded from all directions, resulting in a missing wedge of information. Tomograms conventionally reconstructed with filtered back-projection suffer from noise and strong artifacts due to the missing wedge. Here, we propose a deep-learning approach for simultaneous denoising and missing wedge reconstruction called DeepDeWedge. The algorithm requires no ground truth data and is based on fitting a neural network to the 2D projections using a self-supervised loss. DeepDeWedge is simpler than current state-of-the-art approaches for denoising and missing wedge reconstruction, performs competitively and produces more denoised tomograms with higher overall contrast.
Throughout the history of electron microscopy, ribosomes have served as an ideal subject for imaging and technological development, which in turn has driven our understanding of ribosomal biology. Here, we provide a historical perspective at the intersection of electron microscopy technology development and ribosome biology and reflect on how this technique has shed light on each stage of the life cycle of this dynamic macromolecular machine. With an emphasis on prokaryotic systems, we specifically describe how pairing cryo-EM with clever experimental design, time-resolved techniques, and next-generation heterogeneous structural analysis has afforded insights into the modular nature of assembly, the roles of the many transient biogenesis and translation co-factors, and the subtle variations in structure and function between strains and species. The work concludes with a prospective outlook on the field, highlighting the pivotal role cryogenic electron tomography is playing in adding cellular context to our understanding of ribosomal life cycles, and noting how this exciting technology promises to bridge the gap between cellular and structural biology.
Cryo-electron tomography (cryoET) is a technique that captures images of biological samples at different tilts, preserving their native state as much as possible. Along with the partial tilt series and noise, one of the major challenges in estimating the accurate 3D structure of the sample is the deformations in the images incurred during the acquisition. We model these deformations as continuous operators and estimate the unknown 3D volume using implicit neural representations. This framework allows to easily incorporate the deformation and estimate jointly the deformation parameters and the volume using a standard optimization algorithm. This approach doesn't require training data and can benefit from standard prior in the optimization procedure.
Electron tomography is a widely used technique for 3D structural analysis of nanomaterials, but it can cause damage to samples due to high electron doses and long exposure times. To minimize such damage, researchers often reduce beam exposure by acquiring fewer projections through tilt undersampling. However, this approach can also introduce reconstruction artifacts due to insufficient sampling. Therefore, it is important to determine the optimal number of projections that minimizes both beam exposure and undersampling artifacts for accurate reconstructions of beam-sensitive samples. Current methods for determining this optimal number of projections involve acquiring and post-processing multiple reconstructions with different numbers of projections, which can be time-consuming and requires multiple samples due to sample damage. To improve this process, we propose a protocol that combines golden ratio scanning and quasi-3D reconstruction to estimate the optimal number of projections in real-time during a single acquisition. This protocol was validated using simulated and realistic nanoparticles, and was successfully applied to reconstruct two beam-sensitive metal-organic framework complexes.
Learning compact and interpretable representations of data is a critical challenge in scientific image analysis. Here, we introduce Affinity-VAE, a generative model that enables us to impose our scientific intuition about the similarity of instances in the dataset on the learned representation during training. We demonstrate the utility of the approach in the scientific domain of cryo-electron tomography (cryo-ET) where a significant current challenge is to identify similar molecules within a noisy and low contrast tomographic image volume. This task is distinct from classification in that, at inference time, it is unknown whether an instance is part of the training set or not. We trained affinity-VAE using prior knowledge of protein structure to inform the latent space. Our model is able to create rotationally-invariant, morphologically homogeneous clusters in the latent representation, with improved cluster separation compared to other approaches. It achieves competitive performance on protein classification with the added benefit of disentangling object pose, structural similarity and an interpretable latent representation. In the context of cryo-ET data, affinity-VAE captures the orientation of identified proteins in 3D which can be used as a prior for subsequent scientific experiments. Extracting physical principles from a trained network is of significant importance in scientific imaging where a ground truth training set is not always feasible.
Three-dimensional volumetric imaging of cells allows for in situ visualization, thus preserving contextual insights into cellular processes. Despite recent advances in machine learning methods, morphological analysis of sub-nuclear structures have proven challenging due to both the shallow contrast profile and the technical limitation in feature detection. Here, we present a convolutional neural network, supervised deep learning-based approach which can identify sub-nuclear structures with 90% accuracy. We develop and apply this model to C. elegans gonads imaged using focused ion beam milling combined with scanning electron microscopy resulting in the accurate identification and segmentation of all sub-nuclear structures including entire chromosomes. We discuss in depth the architecture, parameterization, and optimization of the deep learning model, as well as provide evaluation metrics to assess the quality of the network prediction. Lastly, we highlight specific aspects of the model that can be optimized for its broad application to other volumetric imaging data as well as in situ cryo-electron tomography.