Figure 2.

(A) Sequential fluorescence images, at 167-ms intervals, of a single Cy3-F₁ molecule at 20 nM ATP. V, vertically polarized fluorescence; H, horizontally polarized fluorescence. Each image (15 × 30 pixels or 1.5 × 3.0 μm²) was averaged spatially over 3 × 3 pixels. (B) Time courses of spot intensities for V and H in A, median-filtered over eight video frames (0.27 s). (C) Time courses of the polarization, P = (V − H)/(V + H), and total intensity, I = V + H, calculated from B. The fluorophore photobleached at ≈55 s. Dashed lines (a, b, and c) are calculated P for the three orientations in D: P = 0.4 × [sin²(θ + 18°) − cos²(θ + 18°)], where θ = 0°, 120°, and 240°.