Calibration-Independent Atomic Force Microscopy

Atomic Force Microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty. To demonstrate calibration-independent AFM, we have achieved concurrent single-molecule nanomechanical profiling of two different proteins. Using orthogonal fingerprinting strategies to sort individual unfolding events, differences in mechanical unfolding forces can be obtained with a 6-fold improvement in accuracy and a 30-fold increase in throughput. Importantly, the performance of calibration-independent AFM is maintained even when averaging data from multiple, independent experiments.