Fig. 2.

Using stepwise photobleaching to estimate FliM-YPet stoichiometry. (A) Bright-field and sequential TIRF (false-color) images of a tethered FliM-YPet cell rotating around a flagellar motor (red cross on bright-field image and red arrow on TIRF image), in the direction indicated by the white arrow. A second motor is visible at the bottom of the TIRF images, as is a fluorescent spot of lower brightness (Green Arrow). (B) Photobleach trace for the motor indicated by the red arrow in A, showing raw (Blue) and filtered (Red) intensities, with expansion of part of the trace (Inset). (C) Power spectrum following Fourier spectral analysis, indicating a peak in periodicity at ∼1.3 kcounts. Stoichiometry distributions using Gaussian kernel density estimation (width = 3) for (D) tethered, cells (Gray) with Gaussian fit (Black) and (E) immobilized cells (Gray) with combined (Black) and individual (Red) two Gaussian fit. The mean ± SD is shown, with 35 spots analyzed for D and E.