Analysis of constrained septin–GFP fusions using polarized fluorescence microscopy shows that septin rings in A. gossypii are ordered. (A) Raw fluorescence acquired by exciting GFPs in an A. gossypii septin ring with four angles of polarized light in a cell expressing Cdc12-conGFP4. Cell outline is shown in white. Bar, 1 µm. (B) Maps of polarization ratio (pr) and GFP dipole angle (azimuth) were calculated for every camera pixel based on the data in A. The pr across the ring is expressed with the angle as blue lines of a length proportional to the pr, oriented according to the calculated azimuth and overlayed on the sum fluorescence (“pr-scaled” image). Alternatively, the amount and orientation of ordered protein is represented using a color scheme (“spectrum” look-up table in ImageJ) in which each angle is represented as a color whose intensity is the product of the fluorescence and the pr (“pr*fluor-scaled angles” image). Bar, 1 µm. (C) Representation of the “offset” angle (θ), which is the distance counterclockwise from the cell growth axis to the mean azimuth. Two perspectives of an A. gossypii septin ring viewed in the xy or top view (left ring) and the xz or cross section view (right ring).