ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software

Digital video files of motile ciliated ependymal (frequency range 34 - 52 Hz) and respiratory epithelial cells (frequency 3 - 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system.The overall mean difference (+/- s.d.) between the ciliaFA and direct measurement high-speed digital imaging methods was -0.05 +/- 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from -1.99 to 1.49 Hz for respiratory and from -2.55 to 3.25 Hz for ependymal cilia.A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 - 52 Hz) and avoids operator error due to selection bias. We have included free access to the ciliaFA plugin and installation instructions in the Additional Files accompanying this manuscript that other researchers may use.