Analysis of ciliary beat frequencies in hamster oviducal explants.

Abstract

We have developed a simple direct method, requiring minimal manipulation, to measure beat frequencies of the cilia on the external surface of hamster oviducal infundibula in vitro. Two perfusion chambers (closed and open) were used; both can be hand-made in a few minutes and discarded after use. Ciliary beat frequencies were determined by measuring variations in light intensity with time in a single pixel positioned over a video image of the beating cilia. Data files were collected using Image 1 software and later transferred to PSI Plot or Lotus 123 spreadsheets for analysis by counting the number of brightness peaks recorded per second or by subjecting the data to Fourier transformation with or without smoothing. These methods of analysis gave similar results. To verify that Image 1 data files contain accurate representations of CBF, videotapes of beating cilia were made and subjected to frame-by-frame analysis. Image 1 interfaced with a standard video camera was found to collect reliable data over a beat frequency range of 0-15 cycles/sec. In some Fourier transforms, secondary peaks were observed and were shown to represent cilia beating at more than one frequency in a sampled region. Coefficients of variation for repeated measurements taken on the same region varied from 4.1% to 9.0%. Small but significant differences were found between beat frequencies at different regions of the same oviduct. When chambers were perfused discontinuously and measurements of beat frequency were made at least 5 min after each perfusion, no effect of perfusion on frequencies was observed. However, during continual perfusion of the open chamber, a slight but significant increase in beat frequency was observed after perfusion was initiated. Muscle contraction, which sometimes occurs in the open chamber, did not affect beat frequency measurements. Infundibula could be stored at 4 degrees C overnight without any negative effect on beat frequencies. Cold storage also reduced muscle contraction. Placement of a small coverslip on infundibula in the open chambers was also found to reduce muscle contraction and facilitate beat frequency measurements. Coverslipping did not affect beat frequencies. This method of beat frequency analysis will be valuable for analyzing factors that regulate or influence cilia in mammalian oviducts.