Fig. 6.
Counting the number of FliM-Dendra2 molecules in the flagellar motor expressed in ΔfliM strain. (A) Bright-field and (B) FliM-Dendra2 PALM overlay image show that the motor proteins mainly localize as clusters at the cell membrane. Clusters located at the lateral periphery of the cells (arrow head) were selected for the molecular counting, with the exclusion of clusters that were elongated (chevron), located at the cell pole (chevron), or off the periphery (arrow), and surrounded by dispersed molecules (two-headed arrow). (Scale bar, 500 nm.) (C) Diffraction-limited FliM-Dendra2 image. Dashed line, contour of the cell. (D) Iterative optimal-τc counting method estimates 33 ± 1 (mean ± SEM) FliM-Dendra2 molecules per cluster, which is similar to the previously quantified number of 34 molecules per flagellar motor. In contrast, the blinking noncorrected method (τc = 0) estimates 40 ± 1. A total of 89 clusters were analyzed.