Fast single molecule localization microscopy in 3D
- 1University of Cambridge
Abstract
Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.
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BibTeX
@misc{allgeyer2026fast,
title = {{Fast single molecule localization microscopy in 3D}},
author = {E. Allgeyer and J. H. Richens and J. Lefevere-Laoide and E. K. Paluch and G. Sirinakis and D. St Johnston},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.29.755394},
url = {https://www.biorxiv.org/content/10.64898/2026.09.29.755394v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Fast single molecule localization microscopy in 3D
AU - E. Allgeyer
AU - J. H. Richens
AU - J. Lefevere-Laoide
AU - E. K. Paluch
AU - G. Sirinakis
AU - D. St Johnston
PY - 2026
DA - 2026/09/29
PB - bioRxiv
DO - 10.64898/2026.09.29.755394
UR - https://www.biorxiv.org/content/10.64898/2026.09.29.755394v2
AB - Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.
ER -