whole-cell-super-resolution-imaging-via-dna-paint-on-spinning-disk

Whole-cell Super-Resolution Imaging <em>via</em> DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Single-molecule localization microscopy (SMLM) enables nanoscale imaging of cellular structures but is typically restricted to imaging near the coverslip ...

University College London

Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein-protein association () and dissociation () constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.

Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy.

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.

Article	Year
Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy. Nano letters\| PubMed ID: 39432814	2024
Super-resolution imaging in whole cells and tissues via DNA-PAINT on a spinning disk confocal with optical photon reassignment. Nature communications\| PubMed ID: 40442066	2025

Olivia P. L. Dalby

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