Noninvasive protein structural flexibility mapping by bimodal dynamic force microscopy.

Mapping of the protein structural flexibility with sub-2-nm spatial resolution in liquid is achieved by combining bimodal excitation and frequency modulation force microscopy. The excitation of two cantilever eigenmodes in dynamic force microscopy enables the separation between topography and flexibility mapping. We have measured variations of the elastic modulus in a single antibody pentamer from 8 to 18 MPa when the probe is moved from the end of the protein arm to the central protrusion. Bimodal dynamic force microscopy enables us to perform the measurements under very small repulsive loads (30-40 pN).

Bimodal atomic force microscopy imaging of isolated antibodies in air and liquids.

We have developed a dynamic atomic force microscopy (AFM) method based on the simultaneous excitation of the first two flexural modes of the cantilever. The instrument, called a bimodal atomic force microscope, allows us to resolve the structural components of antibodies in both monomer and pentameric forms. The instrument operates in both high and low quality factor environments, i.e., air and liquids. We show that under the same experimental conditions, bimodal AFM is more sensitive to compositional changes than amplitude modulation AFM. By using theoretical and numerical methods, we study the material contrast sensitivity as well as the forces applied on the sample during bimodal AFM operation.