Schematics of relevant frames of reference and polarized intensities. (A) Definitions of the orientation of the vector representing the rhodamine probe absorption and emission dipoles, β_P, α_P, in the Actin Frame (1; x_A, y_A, z_A) and θ_P, ϕ_P, in the Lever Frame (2; x_L, y_L, z_L), which is itself shown, β_L, α_L, in the Actin Frame (3). (B) Laboratory frame: z_laboratory is aligned in the optical axis toward the microscope objective; x_laboratory and y_laboratory are parallel to the slide surface. Note that the z_laboratory and y_A axes, which are identical, point downward (Materials and methods) but are drawn pointing upward in A for clarity. Experimental setup for polTIRF microscopy: A 532-nm laser is initially split into paths 1 and 2 and is polarized using an optical setup similar to the one described in Beausang et al. (2008a). At the sample, the two beams in orthogonal planes, x_laboratory–z_laboratory and y_laboratory–z_laboratory, alternatingly strike the quartz slide at angles (measured from the optical axis) slightly larger than the critical angle required for TIRF (Hecht, 2001). Each beam is sequentially polarized by Pockels cells into four polarizations: (1) horizontally (s1 and s2, perpendicular to the x_laboratory–z_laboratory and y_laboratory–z_laboratory scattering planes for paths 1 and 2, respectively); (2) in the scattering planes (p1 and p2, respectively); and (3 and 4) ±45° (L1/R1 and L2/R2, at ±45° angles with respect to the scattering plane). 12- and 16-channel measurements: Emission from the excited sample is split into its x_laboratory and y_laboratory components for detection by avalanche photodiodes (Materials and methods), leading to a maximum of 16 unique combinations of the eight time-multiplexed input paths and polarizations and two simultaneously detected polarized emission intensities. 12-channel measurements were made when the L and R polarizations were not used in path 2.