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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663051PMC
http://dx.doi.org/10.1021/acs.nanolett.5b03166DOI Listing

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