Multiparametric Atomic Force Microscopy Imaging of Biomolecular and Cellular Systems

There is a need in biochemical research for new tools that can image and manipulate biomolecular and cellular systems at the nanoscale. During the past decades, there has been tremendous progress in developing atomic force microscopy (AFM) techniques to analyze biosystems, down to the single-molecule level. Force distance (FD) curve-based AFM in particular has enabled researchers to map and quantify biophysical properties and biomolecular interactions on a wide variety of specimens. Despite its great potential, this AFM method has long been limited by its low spatial and temporal resolutions. Recently, novel FD-based multiparametric imaging modalities have been developed, allowing us to simultaneously image the structure, elasticity and interactions of biological samples at high spatiotemporal resolution. By oscillating the AFM tip, spatially resolved FD curves are obtained at much higher frequency than before, and as a result, samples are mapped at a speed similar to that of conventional topographic imaging. In this Account, we discuss the general principle of multiparametric AFM imaging and we provide a snapshot of recent studies showing how this new technology has been applied to, biological specimens, from soluble proteins to membranes and cells. We emphasize novel methodologies that we recently developed, in which multiparametric imaging is combined with probes functionalized with chemical groups, ligands, or even live cells, in order to image and quantify receptor interaction forces and free-energy landscapes in a way not possible before. Key breakthroughs include observing the mechanical and chemical properties of single proteins in purple membranes, measuring the electrostatic potential of transmembrane pore forming proteins, structurally localizing chemical groups of water-soluble proteins, mapping and nanomechanical analysis of single sensors on yeast cells, imaging the sites of assembly and extrusion of single filamentous bacteriophages in living bacteria, unravelling the adhesive properties of biofilm-forming microbial pathogens, mapping the ligand-binding free energy landscape of human membrane receptors in proteoliposomes, and finally, the nanomechanical mapping of the first binding events of viruses to animal cells. In the coming years, it is anticipated that multiparametric AFM imaging will be increasingly used by chemists from broad horizons, enabling them to shed light into the sophisticated functions of biomolecular and cellular systems.