Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM
Simone Benaglia, Victor G. Gisbert, Alma P. Perrino, Carlos A. Amo & Ricardo Garcia
Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip–sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.
a, Scheme of the cantilever deflection in bimodal AFM. The deflection signal has low- and high-frequency components. The low-frequency component coincides with the resonant frequency of the first mode of the cantilever, whereas the high-frequency component coincides with the resonant frequency of the second mode. In the right panel, blue represents the total deflection of the cantilever, red the deflection of the first mode and orange the deflection of the second mode. b, Scheme of the feedback loops in bimodal AM–FM. The feedback to generate the topography controls the amplitude of the first mode. Two feedbacks act on the parameters of the second mode. One keeps the phase shift at 90° with respect to the driving force; the other keeps the amplitude A2 at a fixed value (Asp2). The last step is achieved by varying the driving force of the second mode. c, The transformation of bimodal data into nanomechanical properties requires the use of a contact mechanics model. Adapted from ref. 16 (https://pubs.acs.org/doi/10.1021/acsnano.7b04381); further permissions related to the material excerpted should be directed to the American Chemical Society.