JoVE Video Dataset

Super-resolution imaging has revolutionized biological research by revealing structural details at the nanoscale. Most optical super-resolution methods rely on fluorescent labeling of biomolecules, which enables specific tagging of molecular species but can disrupt cellular functions, introduce inaccuracies from linker molecules, and fail to provide the consistent high labeling density required for chromatin imaging at the scale of individual DNA molecules. A technology that enables label-free, in situ genomic imaging with nanometer resolution would profoundly impact biology. Here, we present a protocol that harnesses the intrinsic fluorescence of DNA to perform spectroscopic single-molecule localization microscopy (sSMLM). The protocol details sample preparation, data acquisition, and spectral analysis. Briefly, a thin DNA gel is created by depositing a polynucleotide solution onto glass and allowing it to dry for hours. After gel formation, the sample is imaged in the presence of an imaging buffer using sSMLM. The recorded dataset comprises a zeroth-order image, providing spatial localizations, and a first-order image, encoding the emission spectrum of each localization. Spatial reconstructions are generated from the zeroth-order data, after which the corresponding spectra are extracted from the first-order signal. Finally, we demonstrate the feasibility of this approach using multiple excitation wavelengths and DNA molecules with varying lengths, sequences, and compositions.