Visualizing individual Cy3-ATP molecules binding and releasing from myofibrils. (a) Using fluorescently tagged ATP (Cy3-ATP; untreated: n_events = 8,478, n_myofibrils = 9) microscopic imaging of individual myofibrils was performed on a custom-built imaging system where the illumination laser path is offset in the back focal plane to generate an oblique angle illumination field (OAF). Also shown is the structure of the sarcomere, where the D, C, and P zones are shaded in only one half sarcomere; this structure is also present on the other side of the M-line. (b) This system provides images of ATP binding and release events (Video 1) that are tracked using TrackMate (ImageJ). Individually bound ATP molecules are shown in magenta in the image. Scale bar indicates 2 µm. (c) All lifetimes are plotted as a cumulative residence time histogram and fitted to three exponentials (residuals shown in the inset). (d) The resulting amplitudes correspond to the different myosin states are normalized according to their rate constant (see Materials and methods).