Force Spectroscopy with 9-μs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry

Abstract

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.

High-speed force spectroscopy: microsecond force measurements using ultrashort cantilevers

“…Commercially available ultrashort HS-AFM cantilevers (Table 1) are not optimal for force measurements. While they provide excellent time resolution (sub-μs), ultrashort cantilevers may present limited force precision performance and “ringing” in the force response (Edwards et al 2017). In addition, close proximity to the substrate leads to an important increase in the effective viscous drag, which may limit fast loading velocities.…”

“…FIB milling has been employed to improve the force stability and precision while maintaining an excellent temporal resolution. The group of Perkins has modified and tested different milling strategies on commercially available cantilevers to decrease the stiffness and the hydrodynamic drag coefficient (Edwards et al 2017, 2015; Bull et al 2014; Edwards and Perkins 2017). Indeed, an important assumption in single molecule force spectroscopy (SMFS) analysis methods is the use of overdamped force probes ( Q < 0.5) (Bell 1978; Dudko et al 2006; Evans and Ritchie 1997; Merkel et al 1999).…”

FEATHER: Automated Analysis of Force Spectroscopy Unbinding and Unfolding Data via a Bayesian Algorithm

“…We note that the data sets used here were acquired with a relatively long, soft cantilever. Recent efforts in improving the precision and time resolution of AFM cantilevers will immediately aid FEATHER's ability to detect smaller and more closely spaced unfolding intermediates (22,23). By predicting BCC compares the degree of overlap between the human-annotated and algorithmically predicted two-dimensional distribution of rupture force versus loading rate (e.g., Fig.…”

Meeting report – NSF-sponsored workshop ‘Progress and Prospects of Single-Molecule Force Spectroscopy in Biological and Chemical Sciences’

“…Thomas Perkins (JILA/NIST and the University of Colorado, Boulder, CO) presented recent technical developments that have greatly increased the time resolution (by about 100-fold) and force precision (by about 10-fold) of AFM-based SMFS (Edwards et al, 2017). This increase in performance was demonstrated in pulling experiments on individual bacteriorhodopsin (bR) molecules embedded in their native lipid bilayer, which captured unfolding intermediates in unprecedented detail, including those separated by as few as three amino acids (Yu et al, 2017).…”