Turnkey video-rate atomic force microscopy for nanometre resolution imaging of functional biomolecules and cellular surfaces

Microscopes are our windows to the cell. Their importance is illustrated by the recent awards of Nobel prizes for super-resolution fluorescence microscopy (2014) and for cryo-electron microscopy (2017). These Nobel prizes also highlight the main types of microscopy that are currently used in biology: fluorescence microscopy, which can provide information on live cells, but at relatively poor resolution (~100s of nm), and electron microscopy, which can provide high-resolution images of biological molecules and cells, but requires samples to be frozen. Using atomic force microscopy, we circumvent some of the limitations of other types of microscopy by effectively tracing the surface of a sample (biological or other) with a very sharp needle, enabling us to image functional biological molecules and living cells as ~1 nm resolution. Since we trace these samples line by line to build an entire image, this is a relatively slow type of microscopy.

Here we apply for funding to purchase a so-called high-speed atomic force microscope, which incorporates multiple developments to speed image acquisition up from minutes to less than seconds per frame. We will install the equipment in a well-established user facility and anticipate wide use across different disciplines and institutions. For the short-to-medium term, first experiments we plan are related to protein-DNA interactions that determine DNA packing in the cell, commercial DNA-based nanomaterials developed to facilitate chemical reactions, reshaping of biological membranes, molecular interactions that define how living cells bind to substrates, disruption of bacterial surfaces by next-generation antibiotics, and the molecular structures that define cell shape and mechanics.

High-speed atomic force microscopy (HS-AFM) provides us with a tool to visualise a wide range of biological processes at nanometre resolution in real time. Compared with conventional AFM, HS-AFM has improved the temporal resolution at which images can be recorded from the minute to the (sub)second time scale. This has enabled us, for example, to view the stepping of myosin V motors, the formation of ESCRT spirals, the assembly of immune pores, and the opening and closing of structural-maintenance-of-chromosomes complexes. Such data aid us to understand the pathways by which biomolecular processes occur and to identify kinetic bottlenecks, thus providing molecular-scale understanding that is hard to achieve by other methods.

Here we propose to purchase user-friendly HS-AFM equipment and its installation in a well-established and easily accessible AFM user facility at the London Centre for Nanotechnology, aiming provide low-threshold access to HS-AFM to a wide range of users from academia and industry, working in disciplines including single-molecule biophysics, synthetic biology/ biomaterials, and molecular, structural, micro- and cell biology. To facilitate such wide application, we have identified a set of initial projects in these various remits, which are all to benefit from access to HS-AFM as proposed here.