In order to investigate DNA/chromatin structure, one may employ optical microscopy as a method of choice, due to feasibility and availability. However, conventional light optical imaging approaches suffer from diffraction of the visible wavelengths used, preventing the resolution of structures that are spaced closer than about 200 nm laterally and 600 nm axially. On the other hand, many other approaches, in particular Electron Microscopy and Electron Spectroscopy Imaging, provided a great amount of knowledge on chromatin nanostructures, down to the few nanometer resolution range. However, these important advantages are balanced by a number of drawbacks, such as the time-consuming sample preparation and the harsh preparation conditions which are likely to alter the structure of interest. To overcome this dilemma, recently novel visible light ‘superresolution’/’nanoscopy’ imaging approaches have emerged. Presently especially structured illumination microscopy and Single Molecule Localisation Microscopy (SMLM) have been established as useful approaches [1]. Using fluorescence-based SMLM methods, it was shown that one may use DNA basepair analogs labelled with special fluorophores, ubiquitous H2B histone targeting, or cyanine derivated DNA dyes in order to better resolve chromatin in the eukaryotic cell nucleus or DNA fibers and bacteria. However, no SMLM studies were performed so far on single cell nuclei with directly stained DNA. Here we present novel application of specific DNA dyes in order to obtain nuclear DNA density maps with high optical and structural resolution. For this, we applied a special SMLM variant, Spectral Precision Distance Microscopy (SPDM) [2]. In mammalian cell nuclei, this SPDM technique yielded a single molecule localisation precision in the order of 15 - 30 nm, corresponding to an optical (two-point) resolution of roughly 40 - 70 nm. We investigated various DNA structures and obtained data with a single DNA fluorophore density as high as 5000/µm2. This constitutes a significant improvement in comparison to previously obtained SPDM data for H2B-GFP histones (100 - 400/µm2). We anticipate that in the near future, this approach may contribute to obtain a three dimensional DNA mapping of genome nanostructures of various cell types, such as stem cells and differentiated cells, or normal and cancer cells. Such precise density maps may also serve as a basis for numerical modeling of the nuclear genome.
[1] C. Cremer, B.R. Masters, Resolution enhancement techniques in microscopy, Eur. Phys. J. H. 38 (2013) 281–344.