Significance The advent of high-speed atomic force microscopy (HS-AFM) changed the field of biology considerably. HS-AFM is the only method where in situ dynamics of biological samples and imaging can be coupled with a spatial resolution of 1 to 5 nm in the horizontal direction. Unlike electron or cryo-electron microscopy, HS-AFM does not require fixation or freezing of the samples, and has the ability to derive kinetic parameters by recording the live movements of single-molecule dynamics. In this paper, we used HS-AFM to investigate directly the mechanisms of cardiac muscle activation. We visualized the muscle regulatory tropomyosin–troponin complex movements during activation by calcium or myosin (motor that drives contraction), and the structural transitions that happen during these events.