Super-resolution imaging in whole cells and tissues via DNA-PAINT on a spinning disk confocal with optical photon reassignment

Cecilia Zaza, Megan D. Joseph, Olivia P. L. Dalby, Rhian F. Walther, Karol Kołątaj, Germán Chiarelli, Franck Pichaud, Guillermo P. Acuna and Sabrina Simoncelli ()
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Cecilia Zaza: University College London
Megan D. Joseph: University College London
Olivia P. L. Dalby: University College London
Rhian F. Walther: University College London
Karol Kołątaj: University of Fribourg
Germán Chiarelli: University of Fribourg
Franck Pichaud: University College London
Guillermo P. Acuna: University of Fribourg
Sabrina Simoncelli: University College London

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Single-Molecule Localization Microscopy (SMLM) has traditionally faced challenges to optimize signal-to-noise ratio, penetration depth, field-of-view (FOV), and spatial resolution simultaneously. Here, we show that DNA-PAINT imaging on a Spinning Disk Confocal with Optical Photon Reassignment (SDC-OPR) system overcomes these trade-offs, enabling high-resolution imaging across multiple cellular layers and large FOVs. We demonstrate the system’s capability with DNA origami constructs and biological samples, including nuclear pore complexes, mitochondria, and microtubules, achieving a spatial resolution of 6 nm in the basal plane and sub-10 nm localization precision at depths of 9 µm within a 53 × 53 µm² FOV. Additionally, imaging of the developing Drosophila eye epithelium at depths up to 9 µm with sub-13 nm average localization precision, reveals distinct E-cadherin populations in adherens junctions. Quantitative analysis of Collagen IV deposition in this epithelium indicated an average of 46 ± 27 molecules per secretory vesicle. These results underscore the versatility of DNA-PAINT on an SDC-OPR for advancing super-resolution imaging in complex biological systems.

Date: 2025
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DOI: 10.1038/s41467-025-60263-w

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