Analysis of ependymal ciliary beat pattern and beat frequency using high speed imaging: comparison with the photomultiplier and photodiode methods

BACKGROUND: The aim of this study was to compare beat frequency measurements of ependymal cilia made by digital high speed imaging to those obtained using the photomultiplier and modified photodiode techniques. Using high speed video analysis the relationship of the power and recover strokes was also determined. METHODS: Ciliated strips of ependyma attached to slices from the brain of Wistar rats were incubated at 30°C and observed using a ×50 water immersion lens. Ciliary beat frequency was measured using each of the three techniques: the high speed video, photodiode and photomultiplier. Readings were repeated after 30 minutes incubation at 37°C. Ependymal cilia were observed in slow motion and the precise movement of cilia during the recovery stroke relative to the path travelled during the power stroke was measured. RESULTS: The mean (95% confidence intervals) beat frequencies determined by the high speed video, photomultiplier and photodiode at 30°C were 27.7 (26.6 to 28.8), 25.5 (24.4 to 26.6) and 20.8 (20.4 to 21.3) Hz, respectively. The mean (95% confidence intervals) beat frequencies determined by the high speed video, photomultiplier and photodiode at 37°C were 36.4 (34 to 39.5), 38.4 (36.8 to 39.9) and 18.8 (16.9 to 20.5) Hz. The inter and intra observer reliability for measurement of ciliary beat frequency was 3.8% and 1%, respectively. Ependymal cilia were observed to move in a planar fashion during the power and recovery strokes with a maximum deviation to the right of the midline of 12.1(11.8 to 13.0)° during the power stroke and 12.6(11.6 to 13.6)° to the left of the midline during the recovery stroke. CONCLUSION: The photodiode technique greatly underestimates ciliary beat frequency and should not be used to measure ependymal ciliary beat frequency at the temperatures studied. Ciliary beat frequency from the high speed video and photomultiplier techniques cannot be used interchangeably. Ependymal cilia had minimal deviation to the right side during their power stroke and to the left during the recovery stroke.