Knowing the force response characteristics of the tension sensor module (TSM) is key to a conclusive molecular force measurement. Therefore, we have carefully calibrated our FRET modules (including donor and acceptor fluorophore) by single-molecule force spectroscopy to determine critical molecular properties including folding/unfolding transition pathways, reversibility, and force sensitivity. These experiments demonstrated that our TSMs respond very quickly (in the millisecond to sub-millisecond timescale) to applied mechanical loads, and they quickly refold once forces are reduced. Importantly, the folding/unfolding transitions are hysteresis free, allowing unambiguous FRET–force measurements. At this point, four calibrated biosensors are available that respond to distinct force ranges; please click for detailed information: F40, FL, HP35 and HP35st. This molecular toolbox allows the evaluation of physiologically relevant forces that act across individual molecules in living cells.

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