Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.superresolution fluorescence microscopy | label-free imaging | nucleic acids | chromatin topology | chromosome A dvances in genomics and molecular biology over the past decades revolutionized our knowledge of biological systems. Despite our expanded understanding of biological interactions, there continues to be a limited understanding of these complex molecular processes in nonperturbed, structurally and dynamically complex cellular systems (1). As such, it is of critical importance to develop methods that allow direct visualization of nanoscale structures where these processes take place in their native states. Recently, superresolution fluorescence microscopy techniques, including stimulated emission depletion microscopy, structured illumination microscopy, and photon localization microscopy (PLM), such as photoactivated localization microscopy and stochastic optical reconstruction microscopy (STORM), have extended the ultimate resolving power of optical microscopy far beyond the diffraction limit (2-6), facilitating access to the organization of cells at the nanoscale by optical means. Although superresolution imaging of biological structures using labeled proteins has been well documented due to a wide range of methodologies that provide desirable labeling properties (7,8), and despite tremendous interest and demonstrated need, there are few nanoscopic methods to image macromolecular structures formed by nucleic acids due to constraints in labeling (9-14). Likewise, the limited techniques that currently exist cannot ...