The approach, described by Ahmed H. Shaib, Mahmoud M. Alawieh and Stefan O. Rizzoli in Nature Protocols, combines one-step nanoscale expansion microscopy with fluctuation-based super-resolution analysis. The resulting workflow, called ONE microscopy, is intended to make molecular-scale imaging more accessible to laboratories that do not have access to cryo-electron microscopes or specialized high-end optical systems.

Traditional light microscopy is limited by diffraction, a physical effect that causes light from two nearby objects to blur together when they are too close. Even modern super-resolution methods can require complex instruments, intense labeling strategies or extensive computational processing. Expansion microscopy takes a different route. Researchers anchor biological molecules within a swellable polymer network and then expand the material, increasing the physical distance between fluorescent labels.

The expansion step does not automatically reveal every molecular detail. A protein’s structure can still be represented by only a small number of fluorescent signals, and conventional images may contain noise, background fluorescence and motion-related fluctuations. ONE microscopy addresses these limitations by analyzing changes in fluorescence intensity over time. These fluctuations contain information about the presence, position and behavior of labeled molecules that may not be obvious in a single frame.

In practical terms, the method links chemical preparation, physical enlargement and computational analysis into a single workflow. Samples are embedded in a polymer gel, labeled to identify the proteins of interest and then expanded. After expansion, researchers acquire image sequences using conventional fluorescence equipment. Specialized software analyzes the temporal variation in the recorded signals, extracting spatial information beyond what a standard diffraction-limited image would provide.

This combination is significant because it shifts the focus from simply locating a protein to examining its overall shape. For many biological questions, knowing that a protein is present is not enough. Its size, orientation and structural organization can determine how it interacts with membranes, vesicles, organelles or neighboring proteins. Directly observing those features could help researchers investigate molecular machines in their native cellular environments rather than relying exclusively on purified samples or averaged structural models.

The protocol is designed to work across a broad range of biological materials, including purified proteins, cultured cells and tissues. That flexibility could make the technique useful for researchers studying protein organization at multiple scales. A purified protein might provide a controlled test of shape reconstruction, while cells and tissues could reveal how the same protein is arranged amid the crowded and complex environment of living biology.

A major part of the reported advance is the accompanying software package. Fluctuation-based imaging can be powerful, but its usefulness depends on reliable data processing, and computational analysis has often been a barrier for non-specialist users. The authors present the software as stable and user-friendly, with the goal of making the analysis more efficient, reproducible and practical for laboratories using standard fluorescence microscopes.

ONE microscopy does not replace cryo-electron microscopy, which remains capable of resolving structures at atomic or near-atomic scales under appropriate conditions. Nor does it eliminate the challenges associated with labeling, gel chemistry, image quality and sample preparation. Expansion can introduce distortions, and successful imaging depends on preserving the relationship between the fluorescent labels and the underlying structures. Nevertheless, the method offers a complementary strategy: rather than averaging thousands or millions of molecules, it aims to examine individual protein shapes through expanded, fluorescence-labeled specimens.

The researchers describe the workflow as a practical framework for protein imaging on conventional equipment. By combining physical separation of fluorophores with information extracted from fluorescence fluctuations, the method brings nanoscale structural analysis closer to routine biological imaging. Its broader impact may come not from replacing existing forms of super-resolution, but from making a previously specialized capability more reproducible and attainable for laboratories investigating how individual proteins operate inside cells and tissues.

Subject of Research: One-step nanoscale expansion microscopy for visualizing individual protein shapes using conventional fluorescence microscopes.

Article Title: ONE microscopy.

Article References: Shaib, A.H., Alawieh, M.M. & Rizzoli, S.O. ONE microscopy. Nature Protocols (2026). https://doi.org/10.1038/s41596-026-01399-x

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41596-026-01399-x

Keywords: expansion microscopy, ExM, nanoscale imaging, super-resolution microscopy, fluctuation-based analysis, protein structure, fluorescence microscopy, single-protein imaging, cryo-electron microscopy, biological imaging

The study meticulously characterizes a distinct BAM complex that diverges significantly from the canonical architecture well-studied in Proteobacteria like Escherichia coli. Utilizing state-of-the-art cryo-electron microscopy (cryo-EM), the team resolved the complex’s structure at near-atomic resolution. This advance allowed for the first visualization of structural proteins comprising the BAM in Bacteroidota, revealing unique adaptations that reflect the evolutionary trajectory and ecological niches of these bacteria.

Specifically, the BAM complex in Bacteroidota comprises a β-barrel protein scaffolded by auxiliary lipoproteins that differ both in sequence and structure from their Proteobacterial counterparts. The central component forms a stable β-barrel channel essential for guiding nascent OMPs into the outer membrane. Nonetheless, what stands out is the presence of novel protein domains that appear to modulate substrate recognition and insertion, suggesting functional specialization. These adaptations may contribute to the unique outer membrane properties critical for Bacteroidota’s environmental resilience and interactions within host microbiomes.

Technically, this revelation shifts our understanding of BAM’s evolutionary plasticity. While previous models depicted a relatively conserved assembly mechanism among Gram-negative bacteria, the current data highlight that Bacteroidota BAM operates via distinct molecular interfaces and conformational dynamics. The discovery raises compelling questions about how these structural differences influence BAM’s efficiency, substrate specificity, and response to stress or antimicrobial agents.

The research taps into complex biophysical techniques beyond cryo-EM, integrating cross-linking mass spectrometry and molecular dynamics simulations to map the inter-protein contacts and dynamic behavior under physiological conditions. Such a multidisciplinary approach underscores the nuanced interplay between protein architecture and function and serves as a blueprint for studying membrane complexes that have hitherto evaded structural characterization.

Furthermore, by resolving the BAM architecture in Bacteroidota, the study provides a fresh lens on bacterial envelope biogenesis, a process vital for nutrient acquisition, signaling, and immune evasion. Unlike Proteobacteria, Bacteroidota often dominate human gut ecosystems where their outer membrane composition affects host health and disease states. Deciphering BAM’s structural specifics hence holds translational potential for modulating microbiome functions and combating infections.

Insights from the study suggest potential avenues for novel therapeutics targeting BAM unique to Bacteroidota. Existing antibiotics rarely exploit species-specific BAM differences because of the presumed conservation across bacteria. Now, it becomes conceivable to develop inhibitors that disrupt BAM assembly only in Bacteroidota pathogens or dysbiotic strains, minimizing collateral damage to beneficial microbiota and reducing resistance pressures.

Beyond clinical implications, this structural elucidation enriches our fundamental understanding of membrane protein biogenesis under evolutionary constraints. Bacteroidota’s divergence in BAM complexity may reflect adaptation to distinct protein substrates or membrane lipid compositions, prompting re-evaluation of models and assumptions entrenched in microbiology textbooks. This exemplifies how bacterial diversity continuously challenges and refines canonical biochemical pathways.

Interestingly, the study also hints at potential co-evolutionary relationships between BAM proteins and their outer membrane substrates, indicated by co-variation in specific interaction motifs. This co-evolution likely drives the functional specialization observed and presents an attractive target for computational antisense or peptide-based design strategies investigating antimicrobial intervention points.

The utilization of cryo-EM represents a technological tour de force in microbial structural biology. Achieving the high resolution needed to dissect the BAM complex required significant optimization of sample preparation, including lipid environment mimetics and cryo-protection procedures. Such methodological advances portend a new era where complex membrane protein machineries in diverse bacteria will be structurally accessible, accelerating discovery.

This study also provides a comparative framework to investigate how other understudied bacterial phyla assemble their outer membranes. By setting a precedent for deconstructing BAM variability, future research can build a comprehensive map of β-barrel assembly systems across bacterial diversity, deepening evolutionary insights and expanding the molecular toolbox available for biotechnological and medical exploitation.

Moreover, uncovering the structural details of the distinct BAM complex in Bacteroidota feeds into the larger narrative of bacterial adaptability and robustness. The outer membrane serves as a critical barrier and interface, and its assembly is tightly regulated and sophisticated. Such studies illuminate how bacteria tailor these systems to thrive in multifaceted environments, ranging from soil and aquatic ecosystems to complex symbioses within human hosts.

Taking a broader perspective, this work exemplifies the power of interdisciplinary science, merging microbiology, structural biology, computational modeling, and biochemistry to unravel biological complexity. Its success highlights the importance of integrating diverse expertise and cutting-edge technologies to solve long-standing mysteries regarding bacterial physiology and membrane dynamics.

As microbial resistance increasingly threatens public health, detailed structural and mechanistic knowledge like this will be invaluable for next-generation drug discovery efforts. BAM complexes serve as prime antibiotic targets due to their essential roles, and discerning their unique variants across bacterial phyla opens the door to precision antimicrobial therapies, a critical advancement in the fight against resistant pathogens.

In conclusion, the revelation of a structurally distinct β-barrel assembly machinery complex in the Bacteroidota challenges conventional wisdom, enriches our molecular understanding, and offers promising translational opportunities. Future investigations will undoubtedly refine these findings, probe BAM’s functional dynamics in live cells, and harness this knowledge to innovate antimicrobial strategies that are desperately needed in the era of rising antibiotic resistance.

This discovery not only adds a vital piece to the puzzle of bacterial membrane biology but also exemplifies the continuous evolution of scientific knowledge, driven by technological innovation and curiosity. As research in this direction accelerates, it will inspire a new wave of studies aiming to decipher the vast molecular diversity that underpins life at the microscopic scale.

Subject of Research:
Structural biology of the β-barrel assembly machinery (BAM) complex in Bacteroidota bacteria.

Article Title:
Structure of a distinct β-barrel assembly machinery complex in the Bacteroidota.

Article References:
Silale, A., Madej, M., Mikruta, K. et al. Structure of a distinct β-barrel assembly machinery complex in the Bacteroidota. Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-02132-2

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AI Generated