FIGURE 7.

Orthogonal intensities (I_⊥, dark shaded; I_‖, light shaded) and parallel anisotropy (black) of muscle fiber containing Rh-RLC. To adjust anisotropy values to the same scale as polarized intensities, it is plotted as R_‖(t) = [(_‖I_⊥(t) − _‖I_‖(t))/(_‖I_⊥(t) + 2_‖I_‖(t))] × 256 + 128, where _‖I_⊥(t) and _‖I_‖(t)) are the instantaneous fluorescence intensities at time t. Thus the absolute anisotropy r = (R − 128)/256, where R is the measured anisotropy. In A and B they are −0.148 and −0.031 for 1P and 2P, respectively, before the UV flash. However, the absolute values are not reliable because of different sensitivities of the two orthogonal photomultipliers and because of high numerical aperture of the objective. Moreover, the direction of anisotropy change depends on the relative value of the two polarized components, which in turn depends on the sensitivity of the two photomultipliers. In these experiments, the sensitivity was not normalized, so the direction of change, in contrast to the kinetics, is meaningless. (A) 1P signal; (B) 2P signal.