Science and technology at the nano-scale has benefited greatly from the atomic force microscope (AFM), which provides images by measuring the deflection of a probe—a very sharp tip attached to a flexible cantilever—as it scans across the surface of a sample. In conventional dynamic force microscopy (the most common form of AFM), a specific frequency is used to both vibrate the cantilever and measure the tip’s deflection, but information about the sample that is encoded in the nonlinear deflection at other frequencies is irretrievable.
A solution to this problem is found in multifrequency force microscopy, where the excitation and/or deflection measurement is carried out at two or more frequencies. With acquisition times similar to those for conventional AFM, multifrequency force microscopy has the potential to overcome conventional force microscopes’ limitations in spatial resolution. Recently, R. Garcia and E.T. Herruzo from the Instituto de Microelectrónica de Madrid, Madrid, Spain, have reviewed the development of five different types of multifrequency force microscopy—multiharmonic AFM imaging, bimodal AFM, band excitation, torsional harmonic AFM, and nanomechanical holography—and examined their applications in an article published in the April 1 issue of the online journal Nature Nanotechnology (DOI: 10.1038/NNANO.2012.38).
Multiharmonic AFM imaging is straightforward in that the higher harmonic components generated from conventional dynamic AFM are recorded and plotted. However, it is difficult to detect higher harmonics in air with the forces required for high-resolution imaging, requiring the development of special cantilevers. In liquid, where higher harmonics are easier to detect, a bacterial S-layer with 0.5 nm spatial resolution was imaged, as well as nanoscale mapping of the local stiffness and viscoelastic dissipation in living cells.
By using two excitation frequencies tuned to match two of the flexural eigenmodes of the cantilever, bimodal AFM separates topography from other interactions influencing the tip motion, such as magnetic or electrostatic forces, and is compatible for use in air, liquid, and ultrahigh vacuum. Operating at very low forces (50 pN) in liquid, bimodal AFM was used to obtain non-invasive imaging of isolated proteins.
The aim of band excitation is the acquisition of different dynamic curves while the topography of the surface is recorded. A synthesized digital signal is introduced that spans a continuous band of frequencies, while the response is monitored within the same or even larger frequency band. Although it generates a large amount of data and requires sophisticated controllers, either of which may prevent widespread application, band excitation has probed electromechanical coupling in soft biological systems by distinguishing among damping, Young’s modulus, and electromechanical contributions. Ion diffusion in electrochemical batteries has also been studied with band excitation.
In torsional harmonic AFM, the topographic image is from conventional amplitude modulation AFM, but the tip–surface force is obtained simultaneously by integrating the higher harmonics of the torsional signal. The cantilevers are specially designed so that the tip is offset from the cantilever axis, which is beneficial for creation of torque around the axis of the cantilever and enhancing a large number of higher harmonics needed for an accurate calculation of the time-varying force. Torsional harmonic AFM has revealed fractal dimensionality of cancerous cells that differs substantially from that of normal cells.
By simultaneously exciting the sample and the probe, nanomechanical holography generates images of structures beneath the surface of biological or synthetic materials. The waves that propagate through the sample are scattered by internal structural features, which modify their amplitude and phase shift, and eventually emerge at the sample surface where they influence the tip–surface coupling. An image of the subsurface structure is acquired by plotting the modified phase shift as the probe moves across the sample surface. The inner structure of different cells has been imaged with nanomechanical holography as has nanoparticles inside soft materials.
According to the authors of the review article, “This new field provides a promising framework to improve compositional sensitivity and spatial and time resolution of materials in their native environment and, at the same time, allows properties that are not accessible to conventional force microscopes to be measured.” They said, “Multifrequency AFM methods are conceptually more demanding than conventional AFM methods, but this would seem to be a reasonable price to pay to sustain the impressive development of force microscopy that has been seen over the past 25 years.”