Proteins are dynamic molecular machines having structural flexibility that allows conformational changes.

Current methods for the determination of protein flexibility rely mainly on the measurement of thermal fluctuations and disorder in protein conformations and tend to be experimentally challenging. Moreover, they reflect atomic fluctuations on picoseconds timescales, whereas the large conformational changes in proteins typically happen on micro- to millisecond timescales A group of scientists have successfully determined the flexibility of bacteriorhodopsin a protein that uses the energy in light to move protons across cell membranes at the microsecond timescale by monitoring force-induced deformations across the protein structure with a technique based on atomic force microscopy. They named the technique Microsecond force spectroscopy.

Recently, specially designed torsional harmonic cantilevers (THC) have been developed to perform high speed force spectroscopic measurements while scanning the surface in tapping-mode AFM. Owing to the offset location of the sharp tip the torsional vibrations of this cantilever are sensitive to the forces on the vertically oscillating tip. To enable operation of this method in liquids with forces gentle enough to investigate proteins, the scientists redesigned the cantilever geometry and reduced its flexural and torsional force constants. The design was given the title liquid torsional harmonic cantilever (L-THC).

Vibration spectra recorded in aqueous buffer demonstrate the ability of the torsional mode to enhance harmonic signals in liquids. Multifrequency excitation and detection of cantilever vibrations have proven to improve spatial resolution of imaging in liquid environments.

The advantage of the enhancement of multiple harmonic signals with the L-THC is the ability to recover the tip–sample force waveforms which provide high-speed force–distance curves and allow specific material properties to be measured with high spatial resolution.

The entire period of the tip sample force waveform is approx. 130 micro seconds and the interactions span 20 micro seconds. Furthermore, the waveform exhibits an rms. force noise of approx 10 pN. This represents more than three orders of magnitude improvement in force sensitivity compared to the measurements performed with conventional cantilevers in liquid.

This advance enabled a reduction in the peak interaction forces to allow investigations of various types of proteins without causing them to be denatured. In addition, owing to the microsecond duration of force loading, the mechanical properties derived from these waveforms will reflect molecular behaviour at the microsecond timescale.

Work continues to further enhance this technique and develop even further its application.