HS-AFM imaging of D96N bR mutant. (a,b) HS-AFM images of the cytoplasmic side (a) and the extracellular side (b) of D96N bR mutant (imaging rate, 1 fps) under dark (left panels) and green light illumination (right panels) (λ = 532 nm, 0.5 μW). bR trimers are highlighted by the white triangles. The insets in (a) display respective averaged images of trimers. The light-blue circles in (a) indicate a trefoil, and the green bars in (a) and (b) indicate green light illumination. (c) Decay after flash-illumination of the activated bR molecules observed at different pH values (7, 8, and 9). The exponential decay constants (τ) at pH 7, 8, and 9 are 6.7, 25, and 48 s, respectively. The inset shows the absorbance change at 410 nm (that is, decay of the M-intermediate) after flash illumination of D96N measured at various values of pH (7, 8, or 9). (d) Traces (red, blue, and purple marks) of the centers of mass of bR molecules under dark–illumination cycles are superimposed on the image of D96N in the dark. A bR trimer is encircled with the white triangle, and the positions of the α-helical ends facing the cytoplasmic surface (labeled with A–G) are derived from an atomic model of the unphotolyzed state.