Single-Molecule Digital Imaging with Molecular Resolution using DNA-Paint

Recent advances in super-resolution microscopy have allowed the circumvention of the classical diffraction limit of light, and demonstrated the ability of imaging macromolecular protein complexes with single particle averaging methods. However, the ability to obtain fluorescence images within a single molecular complex that allows the study of molecular heterogeneity and low copy number complexes is still challenging, and remains an important goal for the future development of super-resolution microscopy. We here report early results towards this goal of “single-molecule digital imaging” using DNA-PAINT. We introduced three technical advances, including better localization accuracy by obtaining more photons per binding/blinking event, a novel template-based drift correction algorithm, and wide-range tuning of blinking duty cycles for high-density imaging. We demonstrated our approach to obtain molecular-scale resolution on a synthetically designed DNA origami nanostructure, which is made of closely packed (spaced 5 nm apart) DNA molecules with imaging sites, arranged in a “digital” grid pattern. Using small molecule-based oligonucleotide labeling or genetically encoded tags, our imaging technology could achieve molecular resolution within single-molecule complexes. It could be applied to a wide range of biological and biomedical applications including the analysis of densely packed cell surface receptor clusters, cytoplasmic signaling pathways, or genomic organization and short-range interactions.