Determining the location of the bound Cy3-ATP localizations within thick filament zones. (a) Using fluorescently tagged anti–α-actinin antibodies the Z-discs are imaged. Scale bar indicates 2 µm. For greater accuracy, single-molecule localization is used to determine individual antibody locations, which are then used to delineate the sarcomere with 32 nm precision. (b) Individual Cy3-ATP localizations were superlocalized (precision was 29.2 nm in X and 26.7 nm in Y) using TrackMate and plotted relative to the locations of the Z-discs corrected to the horizontal axis (Fig. S3). Using known EM data, the boundaries of the P-, C-, and D-zones were used to bin bound Cy3-ATP localizations. All localizations outside of these zones were discarded. (c) The localization lifetimes were plotted as cumulative residence times. (d) Fitting these to triple exponential decays provides amplitude data to give the relative populations of each myosin state within each thick filament zone (see Table 1 for values). Significant differences (P < 0.05) between corresponding populations relative to the P- and C-zones are indicated with *. All C-zone populations: active (P = 0.0018), SRX (P = 0.0218), and DRX (P = 0.0001) are significantly different from the P-zone. For the D-zone: active (P = 0.04) and DRX (P = 0.0437) populations are significantly different from the P-zone. The only significant difference between D-zone and the C-zone populations is DRX (P = 0.0003).