Correlative AFM and Super-Resolution STED Analysis of DNA Nanostructures

In the past decades, atomic force microscopy (AFM) has become standard in the high-resolution structural analysis of samples ranging from single molecules to complex macromolecular systems. Unlike other high-resolution imaging techniques, it does not require any specific sample modification (except for surface deposition), therefore the risk of introducing artefacts through sample preparation is reduced. In recent years, there has been an increased demand for and application of novel developments featuring high-speed AFM imaging (>10 fps), quantitative nanomechanical characterization of samples, and advanced feedback imaging modes which tweak the maximum attainable resolution. Furthermore, the examination of specific molecules or features carrying immunochemical information has been made possible by combining AFM with recent developments in optical/fluorescence microscopy, and in doing so, complementing the advantages of both techniques and enabling true correlative microscopy.

In this app note, we successfully used the latest generation JPK NanoWizard ULTRA Speed 2 AFM to study DNA-based nanostructures with sub-molecular resolution in vitro. Furthermore, it was demonstrated that the instrument can be successfully coupled to a 2D compact line super-resolution STED instrument and can non-invasively carry out correlative microscopy with lateral resolution beyond 70 nm. The ability of this setup to study dynamic processes with a temporal resolution of over 1 fps was also demonstrated.

Readers can expect to learn about:

• The ability of the NanoWizard ULTRA Speed instrument to study DNA-based nanostructures with sub-molecular resolution in vitro;
• How the NanoWizard ULTRA Speed instrument can be coupled to a super-resolution STED instrument for non-invasive correlative microscopy with a lateral resolution better than 70 nm; and
• The ability of this configuration to image dynamic processes with a temporal resolution greater than 1 fps.

_{KEYWORDS:    Correlative Microscopy; DNA Nanorulers; DNA origami; High-Resolution Imaging; STED; Super-Resolution Imaging}