Fast single molecule localization microscopy in 3D

Edward S. Allgeyer, Jennifer H. Richens, Jeanne Lefévère-Laoide, Ewa K. Paluch, George Sirinakis, Daniel St Johnston

Posted on: 14 October 2026

Preprint posted on 5 October 2026

Intrinsic3D, a new imaging approach that allows researchers to image entire cells and tissues in 3D at single-molecule resolution, making large-volume super-resolution imaging faster and easier.

Selected by Kanishka Parashar

Categories: biochemistry, bioengineering, biophysics, cancer biology, cell biology, developmental biology

Background

Single-molecule localization microscopy (SMLM) techniques have transformed our ability to visualize protein organization at nanometer resolution. However, expanding these approaches to three dimensions remains challenging. Most 3D SMLM methods depend on engineered point spread functions, extensive calibration procedures, thin samples, or slow serial acquisition of multiple z-planes. These limitations make imaging complete cells and tissues time-consuming and computationally demanding.

In this preprinted work, Allgeyer and colleagues introduce Intrinsic3D, a volumetric imaging strategy that integrates the optical sectioning and rapid scanning capabilities of oblique plane microscopy (OPM) with the molecular precision of SMLM. This approach determines axial position by measuring photon counts obtained from sequential sampling of an emitter as an oblique light sheet scans through the sample. In simple terms, the sample is scanned and individual molecules are detected at multiple depths, identifying their precise 3D positions.

Key Findings

Intrinsic3D enables direct volumetric single-molecule localization.

The volumetric imaging was achieved by using a scanning oblique light sheet to collect multiple measurements of the same blinking fluorophore during the time of a single binding event. By combining lateral localization with photon intensity information across sampling positions, the authors reconstructed emitter coordinates directly in XYZ space without requiring volume stitching or PSF engineering.

High-resolution imaging is maintained throughout large cellular volumes.

Intrinsic3D resolved nuclear pore complexes (NPCs) across whole U2OS nuclei, achieving approximately 24 nm lateral resolution while preserving structural measurements consistent with previous studies. Importantly, imaging performance remained stable across extended imaging depths and volumes.

New biological insights into epithelial junction organization.

Applying Intrinsic3D to endogenous E-cadherin revealed unexpected heterogeneity in the distribution of E-cadherin-containing junctions. By mapping nanoscale membrane organization across entire follicle cell epithelia (Figure 4g,h), the authors found that spot adherens junctions contain higher local concentrations of E-cadherin and frequently cluster near tricellular junctions, regions where three cells meet.

Intrinsic3D enables quantitative analysis of tissue architecture.

Beyond visualization, Intrinsic3D enabled quantitative characterization of epithelial organization. The authors measured E-cadherin distribution across distinct cellular domains (Figure 4c) and quantified membrane separation across hundreds of cell-cell contacts (Figure 4d-j), showing how volumetric super-resolution imaging can generate biologically meaningful nanoscale measurements at tissue scale.

Compatibility with multiplexed super-resolution imaging.

By combining Intrinsic3D with Exchange-PAINT, the authors mapped the spatial relationship between E-cadherin and the tricellular junction protein Gliotactin in late-stage Drosophila follicle cells, highlighting the method’s suitability for future 3D spatial proteomics applications.

Snapshot of preprint figure 4 best illustrates the biological power of Intrinsic3D. Following three-dimensional reconstruction and segmentation of Drosophila follicle cells (Figure 4a-b), the authors quantified E-cadherin distribution across distinct cellular domains (Figure 4c-f) and mapped nanoscale membrane organization throughout the epithelium (Figure 4g-j). These analysis revealed that E-cadherin rich spot adherens junctions are preferentially enriched near tricellular junctions and enabled quantitative measurements of membrane separation across thousands of cell-cell contacts. The figure demonstrates how Intrinsic3D can move beyond high-resolution visualization to quantitative spatial cell biology. Preprint figure was made available under a CC-BY 4.0 International license.

What I like & Importance

What I liked the most about this preprint is the technical simplicity and scalability of the described imaging approach as it avoids complicated axial calibrations and post-acquisition stitching. Additionally, the approach shows strong biological validation in human cell lines and Drosophila tissues. Integration with Exchange-PAINT and other quantitative imaging methods make it an important tool for nanoscale spatial imaging and likely useful for researchers studying tissue architecture, developmental biology, mechanobiology and spatial organization of protein complexes.

Future directions & Questions for the authors

How does Intrinsic3D perform in thicker and more optically heterogeneous tissues or organoids?
While the current work focuses on fixed samples, are there aspects of the workflow that could inspire future live-cell implementations?
How compatible is Intrinsic3D with emerging approaches that combine super-resolution imaging with spatial proteomics or multiplexed molecular profiling?
Given the interesting enrichment of E-cadherin at spot adherens junctions, do you see a direct functional role for this structure in sensing or responding to mechanical forces?

Tags: advanced microscopy, imaging

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Author's response

Responses from Prof. Daniel St Johnston to the questions shared

How does Intrinsic3D perform in thicker and more optically heterogeneous tissues or organoids? So far, we have imaged up to ~15-20 µm into our samples, a depth currently set by the length of the light sheet. Imaging deeper is possible with a longer, lower-NA light sheet, at some cost in optical sectioning, and depths of up to ~40 µm may be achievable in favourable samples. In practice, the achievable depth will depend strongly on sample preparation and tissue properties, such as sample-induced aberrations, scattering and background fluorescence, and we have not systematically investigated these limits. We have not yet imaged any organoids but are keen to try.
While the current work focuses on fixed samples, are there aspects of the workflow that could inspire future live-cell implementations? This approach will be hard to combine with live imaging because one needs tens of thousands of volumes containing sparse blinks to build a 3D super-resolved image. Having said that, there have recently been a few papers that perform live PAINT in 2D using protein probes labelled with fluorescent proteins and it may eventually be possible to do this in 3D.

How compatible is Intrinsic3D with emerging approaches that combine super-resolution imaging with spatial proteomics or multiplexed molecular profiling? Since one can perform sequential DNA-PAINT imaging, one can image as many proteins as one has specific probes for, such as nanobodies. This means that one could in principle localise tens of proteins in 3D, which is getting close to spatial proteomics without the need of mass-spectroscopy. It should also be possible to design probes for specific RNAs

Given the interesting enrichment of E-cadherin at spot adherens junctions, do you see a direct functional role for this structure in sensing or responding to mechanical forces? Yes, this is definitely something we would like to explore further.