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

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.