title: Improved Force Spectroscopy Using Focused-Ion-Beam-Modified Cantilevers.
abstract: Atomic force microscopy (AFM) is widely used in biophysics, including force-spectroscopy studies of protein folding and protein-ligand interactions. The precision of such studies increases with improvements in the underlying quality of the data. Currently, data quality is limited by the mechanical properties of the cantilever when using a modern commercial AFM. The key tradeoff is force stability vs short-term force precision and temporal resolution. Here, we present a method that avoids this compromise: efficient focused-ion-beam (FIB) modification of commercially available cantilevers. Force precision is improved by reducing the cantilever's hydrodynamic drag, and force stability is improved by reducing the cantilever stiffness and by retaining a cantilever's gold coating only at its free end. When applied to a commonly used short cantilever (L=40μm), we achieved sub-pN force precision over 5 decades of bandwidth (0.01-1000Hz) without significantly sacrificing temporal resolution (~75μs). Extending FIB modification to an ultrashort cantilever (L=9μm) also improved force precision and stability, while maintaining 1-μs-scale temporal resolution. Moreover, modifying ultrashort cantilevers also eliminated their inherent underdamped high-frequency motion and thereby avoided applying a rapidly oscillating force across the stretched molecule. Importantly, fabrication of FIB-modified cantilevers is accessible after an initial investment in training. Indeed, undergraduate researchers routinely modify 2-4 cantilevers per hour with the protocol detailed here. Furthermore, this protocol offers the individual user the ability to optimize a cantilever for a particular application. Hence, we expect FIB-modified cantilevers to improve AFM-based studies over broad areas of biophysical research.
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States.
authors.forename: J K
authors.initials: JK
authors.lastname: Faulk
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States.
authors.forename: D T
authors.initials: DT
authors.lastname: Edwards
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States.
authors.forename: M S
authors.initials: MS
authors.lastname: Bull
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, United States; University of Colorado, Boulder, CO, United States. Electronic address: tperkins@jila.colorado.edu.
authors.forename: T T
authors.initials: TT
authors.lastname: Perkins
mesh_terms: D001703:Biophysics / Q000379:methods*; D002138:Calibration; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000648:ultrastructure*
chemical_list: D008024:Ligands; D011506:Proteins
keywords: Atomic force microscopy; Cantilever dynamics; Focused-ion-beam modification; Protein folding; Single-molecule biophysics; Single-molecule force spectroscopy