Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50–1000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using an ultrashort (L = 9 μm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-μm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 μm cantilever into one terminated with a gold-coated, 4 × 4 μm reflective region connected to an uncoated 2-μm-wide central shaft. This "Warhammer" geometry achieved 8.5-μs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM.

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In addition to providing the ability to image on the nanoscale, atomic force microscopy (AFM) has the ability to measure small (pN) forces. This ability has led to new insights into conformational changes in biological molecules; in particular, single-molecule force spectroscopy (SMFS) is a powerful tool to investigate folding in proteins. Ideally, one could observe folding in proteins at time scales in the microsecond range with both short-term force precision and long-term force stability. Recent work [1] has shown that to minimize force drift, one must use a soft AFM cantilever due to instrumental noise. Such soft, long cantilevers have poor temporal resolution. When high temporal resolution is required, one choses a shorter cantilever, which has a higher spring constant ( ∝ −3 ) and hence increased force drift. Recently, it has been shown that by modifying a commercially available cantilever with focused ion beam (FIB) milling, short AFM cantilevers (L~40 μm) can be softened to reduce force drift, without sacrificing temporal resolution. The resulting cantilevers offer state-of-the-art force stability and precision, with temporal resolution ~70 μs [2]. To further improve the performance of modified AFM cantilevers, FIB modification strategies need to be developed to extend the technique to ultra-small cantilevers (L~9 μm) , which offer temporal resolution ~1 μs [3].We present several modification strategies, each of which decrease the stiffness of the resultant cantilevers while retaining fast response times. Additionally, we explore techniques that improve the yield of the process.