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 due to the limitations of total internal reflection fluorescence (TIRF) and highly inclined and optical sheet (HILO) illumination. Here, a protocol that leverages a spinning disk confocal microscope equipped with optical photon reassignment (SDC-OPR) to perform SMLM in whole cells is presented. This method enables high-precision single-molecule imaging throughout the full cell volume using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) without the need for custom optics or complex illumination schemes. The protocol provides a stepwise guide on configuring a commercially available spinning disk microscope (e.g., CSU-W1 SoRA) for SMLM imaging, including acquisition parameters optimized for deep imaging. Sample preparation steps are outlined for labeling intracellular targets with DNA-conjugated probes to perform DNA-PAINT imaging, including strategies for multicolor imaging of several targets. Image acquisition is followed by single-molecule localization and reconstruction using standard software packages. Critical considerations for minimizing background, optimizing lateral resolution, and ensuring imaging quality across the full cell depth are also discussed. This accessible protocol allows researchers to perform deep, whole-cell SMLM using standard confocal equipment, expanding the range of biological questions addressable by single-molecule imaging beyond the near-membrane regions.