Generalized focused-ion-beam milling strategy to tune mechanical properties of AFM cantilevers for single-molecule force spectroscopy studies.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables the characterization of individual biological molecules through the application of mechanical force. The spatiotemporal resolution of such measurements depends greatly on the AFM cantilever that is used, specifically its stiffness, hydrodynamic drag, and material composition. Prior work has shown that focused ion beam (FIB) lithographic modification of small cantilevers can be used to lower the spring constant (and thus force noise) and drift while maintaining a relatively fast time response. Published methods for implementing such optimization rely on specific FIB instruments and cantilever types, limiting broad implementation of these methods to improve SMFS data quality. Here, we show that it is possible to achieve such optimized properties using generalized techniques applicable to a broader array of FIB instruments and starting from new types of cantilevers that are presently commercially available. Modified cantilevers exhibited a 90% reduction in spring constant, sub-pN force drift to tens of seconds, and a time response of ∼25 μs in the liquid environment relevant to biological measurements.