Detection of streptavidin–biotin intermediate metastable states at the single-molecule level using high temporal-resolution atomic force microscopy

) to minimize the force-induced disruption and to provide a chance to explore the system in near-equilibrium. Therein, by utilizing a fast sampling rate for the detection of force by atomic force microscopy (20 μs per data point), several transient states of the system were clearly resolved in our force spectroscopy measurements. These key strategies allow the determination of the states' relative positions and free energy levels along the pulling reaction coordinate for the illustration of an energy landscape of the system.