An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy

[Display omitted] •Universal substrate for high-resolution atomic force microscopy of biomacromolecules.•Based on coated highly oriented pyrolytic graphite.•Imaging of proteins, polysaccharides, DNA and DNA-protein complexes. High-resolution atomic force microscopy (AFM) of biomacromolecules is a valuable method for structural studies in biology. Traditionally, the surfaces used for AFM imaging of individual molecules are limited to mica, graphite, and glass. Because these substrates have certain shortcomings, new or modified surfaces that improve the quality of AFM imaging are highly desirable. Here, we describe an improved substrate for imaging of individual biomacromolecules with high-resolution AFM based on graphite surfaces coated by physical adsorption. We provide a detailed methodology, including the chemical structure, synthesis, characterization and the use of a substance that modifies the surface of freshly cleaved graphite, making it suitable for adsorption and AFM visualization of various biomacromolecules while minimizing spatial distortions. We illustrate the advantages of the modified graphite over regular surfaces with examples of high-resolution single-molecule imaging of proteins, polysaccharides, DNA and DNA-protein complexes. The proposed methodology is easy to use and helps to improve substantially AFM imaging of biomacromolecules of various natures, including flexible and/or unstructured sub-molecular regions that are not seen on other AFM substrates. The proposed technique has the potential to improve the use of AFM in structural biology for visualization and morphometric characterization of macromolecular objects.