New ONE Microscopy Technique Resolves Single Protein Shapes at 1 nm Resolution Using Conventional Light Microscopes

A new imaging method called ONE microscopy—combining tenfold expansion with computational fluctuation analysis—enables the visualization of individual protein conformations at approximately 1 nanometer resolution, using only standard fluorescent dyes and ordinary light microscopes. Published in Nature Biotechnology, the technique has already captured calcium-dependent shape changes in a 17-kDa signaling protein and revealed structural differences in protein aggregates from Parkinson’s disease patients, opening a practical bridge between structural biology and clinical microscopy.

The Resolution Barrier: Why a New Approach Was Needed

For decades, light microscopy has been constrained by the diffraction limit of light, which blurs details smaller than about 200–300 nanometers. While this is sufficient to see cells and some organelles, it cannot resolve the shape of a single protein—typically only 2–10 nanometers in size. Super-resolution techniques such as stimulated emission depletion (STED) microscopy and stochastic optical reconstruction microscopy (STORM) can push below this barrier, but they require specialized lasers, complex optical setups, and often custom fluorophores that complicate sample preparation and limit widespread adoption.

Expansion microscopy (ExM) offered an elegant alternative: by physically embedding biological samples in a swellable hydrogel and expanding them isotropically up to tenfold, otherwise unresolvable structures become large enough to be imaged with conventional optics. However, standard ExM protocols typically achieve a resolution of around 60–70 nm after expansion, which is still far from the single-protein scale. While recent advances have pushed the resolution of ExM to approximately 20–30 nm, reliably reaching sub-10 nm resolution for individual proteins—especially small ones below 20 kDa—remained an elusive goal.

ONE microscopy overcomes this limitation by combining the physical magnification of a tenfold expansion protocol (X10 ExM) with a computational post-processing step called super-resolution radial fluctuations (SRRF). The result is a practical, low-cost method that can resolve the hollow center of a single protein and distinguish different conformational states at about 1 nm resolution.

[IMAGE: Side-by-side comparison showing a diffraction-limited image (blurry blob), a standard expansion microscopy image (slightly better but still indistinct), and a ONE microscopy image of calmodulin (clearly showing the dumbbell shape with two lobes separated by ~2–3 nm). Source: adapted from Nature Biotechnology publication.]

How ONE Microscopy Works: The Synergy of X10 ExM and SRRF

The ONE microscopy workflow is deceptively simple and can be performed in any laboratory equipped with a standard epifluorescence or confocal microscope.

First, samples—whether cultured cells, tissue sections, or purified proteins—are fixed and embedded in a dense polyacrylate hydrogel. The protocol uses the X10 ExM method, which achieves a tenfold linear expansion through a combination of high monomer concentration and a digestion step that homogenizes the gel. Because the expansion is isotropic (uniform in all directions), the relative positions of molecules are preserved, allowing accurate structural reconstruction.

After the gel is fully expanded—turning a 100-micrometer-wide sample into a 1-millimeter-wide one—the target proteins are labeled using conventional fluorophores such as Alexa Fluor 488 or Cy3. No special dyes or blinking buffers are required. The expanded, labeled sample is then placed under a standard microscope, and a short video of several hundred frames is recorded while the fluorophores are illuminated. The key insight is that even under constant illumination, the fluorescence from each molecule exhibits subtle fluctuations due to photoswitching, blinking, or other molecular dynamics. These fluctuations are typically treated as noise, but the SRRF algorithm exploits them.

SRRF, originally developed for live-cell super-resolution, analyzes radial symmetries in the temporal fluctuations across each frame. By computing the local degree of radial coherence in the signal over time, the algorithm reconstructs a single high-resolution image in which individual fluorophores are localized with precision far exceeding the diffraction limit. When applied to a sample already physically expanded tenfold, the effective resolution approaches 1 nm in the original (pre-expansion) sample space. The researchers confirmed this resolution using the well-characterized GABA_A receptor, whose crystal structure (PDB 5OJM) provides a known reference.

Crucially, no modifications to the microscope—no additional lasers, no adaptive optics, no specialized cameras—are required. The only added steps beyond standard immunostaining are the hydrogel embedding, expansion, and running the SRRF software (freely available as an ImageJ plugin).

[IMAGE: Workflow diagram: Step 1 – cells or tissue with target protein; Step 2 – embedding in X10 hydrogel; Step 3 – tenfold isotropic expansion (shown as a scale bar growing from 10 μm to 100 μm); Step 4 – fluorescence imaging of expanded gel under standard microscope; Step 5 – SRRF analysis of 200-frame video yields a final image with ~1 nm resolution. Labels: "Sample," "Hydrogel Embedding," "X10 Expansion," "Wide-field Imaging," "SRRF Reconstruction."]

Validating the Method: Watching Calmodulin Change Shape

To demonstrate the capability of ONE microscopy at the single-protein level, the researchers chose calmodulin—a small, 17-kDa calcium-binding protein that undergoes a well-known conformational change upon binding calcium. In its calcium-free state, calmodulin adopts a relatively closed, compact form. When calcium binds, the protein opens into an extended dumbbell shape, with two globular domains connected by a flexible central helix. The distance between the two lobes changes by approximately 2–3 nm, a difference that is impossible to detect with conventional light microscopy but becomes resolvable at 1 nm resolution.

Using ONE microscopy, the team imaged individual calmodulin molecules in both states. The reconstructed images clearly showed the two lobes as distinct fluorescent spots, and the measured distances between them matched the expected values from known crystal structures. In the absence of calcium, the lobes were close together (closed conformation); after adding calcium, the separation increased (open conformation). This was confirmed by statistical analysis of hundreds of single-molecule measurements, yielding a distribution that shifted by the predicted amount.

This result validates that ONE microscopy can capture conformational dynamics of small proteins at the single-molecule level without averaging over many molecules, which is a fundamental requirement for studying transient structural states or heterogeneous populations. It also means that researchers can now directly visualize how a protein changes shape in response to a stimulus—an ability previously restricted to techniques like cryo-electron microscopy (cryo-EM) or X-ray crystallography, which require high sample concentrations, specialized facilities, and often averaging over millions of molecules.

[IMAGE: Two panels showing ONE microscopy images of individual calmodulin molecules. Left panel: calcium-free condition, showing two closely spaced spots (closed conformation). Right panel: calcium-bound condition, showing two spots separated by ~3 nm (open conformation). A schematic overlay of the dumbbell shape is included for clarity.]

Clinical Applications: Imaging Protein Aggregates in Parkinson’s Disease

Beyond basic science, ONE microscopy has immediate potential for clinical diagnostics. The researchers applied the method to analyze protein aggregates from cerebrospinal fluid (CSF) samples of patients with Parkinson’s disease. Aggregates of alpha-synuclein are a hallmark of Parkinson’s pathology, but their size and morphology can vary widely and may correlate with disease subtype or progression. Traditional light microscopy can only see these aggregates as featureless clumps, while electron microscopy provides high detail but is labor-intensive and incompatible with routine clinical workflows.

With ONE microscopy, the team was able to resolve the fine structure of individual alpha-synuclein aggregates directly from patient CSF. They observed distinct morphological features—including elongated fibrils, spherical oligomers, and ring-like structures—that were not discernible with standard expansion microscopy alone. Moreover, they found that aggregates from different patients exhibited different proportions of these shapes, suggesting that the technique could be used to classify disease phenotypes or monitor therapeutic response.

Because ONE microscopy uses standard fluorescent antibodies, it can be readily adapted to any protein of interest. The ability to visualize single protein aggregates in clinical samples without the need for expensive electron microscopes or cryo-EM facilities makes nanoscale imaging accessible to hospital pathology labs and research institutions with limited budgets.

Implications and Future Directions

ONE microscopy bridges a critical gap between high-resolution structural biology and conventional light microscopy. Cryo-EM and X-ray crystallography can achieve atomic resolution, but they require purified proteins in non-physiological conditions and cannot easily visualize proteins in their native cellular environment. Light microscopy, on the other hand, can image live or fixed cells and tissues, but until now could not resolve individual protein shapes. ONE microscopy provides a practical solution: it works with standard lab equipment and widely available reagents, and it can be applied to thin sections, cultured cells, or even clinical samples.

The ~1 nm resolution achieved is sufficient to distinguish not only conformational changes in small proteins but also the binding of drugs or ligands to single protein molecules. This opens new avenues for drug discovery, where researchers could directly observe whether a candidate compound induces a desired structural change in a target protein. It could also enable the study of post-translational modifications, protein–protein interactions, and membrane protein organization at unprecedented resolution.

The developers note that the current method is limited to fixed (dead) samples, as the expansion process requires chemical crosslinking and digestion. However, future versions might incorporate less invasive gels or live-cell compatible protocols. Additionally, while the resolution is ~1 nm, it is not yet atomic resolution—individual atoms remain invisible. But for many biological questions, knowing the shape and relative positions of protein domains is sufficient.

The combination of X10 expansion and SRRF is not the only approach to achieve nanoscale resolution with light microscopy, but it stands out for its simplicity and accessibility. As the protocol becomes standardized and the software more widely adopted, ONE microscopy could democratize single-protein imaging, allowing any lab with a fluorescence microscope to see details that were previously the exclusive domain of colossal facilities.

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Reference:
Wen, G. et al. One-step nanoscale expansion microscopy reveals single protein conformations and aggregates. Nature Biotechnology (2024). DOI: 10.1038/s41587-024-02494-2

Keywords: nanoscale expansion microscopy, single protein imaging, super-resolution radial fluctuations, ONE microscopy, protein conformational changes, Parkinson disease aggregates