Figure 4—figure supplement 3.

Example cilia analysis process.

(A) Example of a single optical section showing a dorsal cilium from Figure 4C2. Dark green arrow indicates the RF. Light green and red dots indicate the base and tip of the cilium, respectively. (B) A mask drawn over the single cilium to calculate only the frequency and orientation in a specific region of interest. (C) The raw fluorescence trace of the inside of the mask over 25 s video (left) and zoomed in over 1 s (right), corresponding to the highlighted area on the left panel, was used to detect peaks and estimate the ciliary beating frequency by counting the number of oscillations per second. (D) The temporal mean of the time series was calculated to see a temporally smoothed overview of the video. (E) Standard deviation projection over 25 s before temporal smoothing. (F) A principal component analysis was performed to grasp which movements of the cilium could explain the most variability, which appeared in PC1 (left) and PC2 (right). (G) Ciliary orientation calculation. Left: window to calculate the orientation manually. Green dot represents the predetermined cilium center from the polyfit function in MATLAB. Black and red dots represent the manual inputs for the base and tip of the cilium, respectively. Right: The value for the manual calculation of orientation was compared to the orientation output of the two MATLAB functions and the one determined to best explain the data (in this case, a tilt of 52.7°) was kept for further analyses. Scale bar is 2 μm (A).