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<p>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 <i>Drosophila</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月29日

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  • Cecilia Zaza,
  • Megan D. Joseph,
  • Olivia P. L. Dalby,
  • Rhian F. Walther,
  • Karol Kołątaj,
  • Germán Chiarelli,
  • Franck Pichaud,
  • Guillermo P. Acuna,
  • Sabrina Simoncelli

摘要

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.