title: Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope.
abstract: 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.
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Devin T
authors.initials: DT
authors.lastname: Edwards
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Jaevyn K
authors.initials: JK
authors.lastname: Faulk
authors.affiliation: Quantum Electronics and Photonics Division, National Institute of Standards and Technology , Boulder, Colorado 80305, United States.
authors.forename: Aric W
authors.initials: AW
authors.lastname: Sanders
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Bull
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Walder
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Marc-Andre
authors.initials: MA
authors.lastname: LeBlanc
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Marcelo C
authors.initials: MC
authors.lastname: Sousa
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.|Department of Molecular, Cellular, and Developmental Biology, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Thomas T
authors.initials: TT
authors.lastname: Perkins
mesh_terms: D018625:Microscopy, Atomic Force / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*
keywords: AFM; atomic force microscopy; cantilever dynamics; focused-ion-beam milling; protein folding; single-molecule biophysics; single-molecule force spectroscopy