Ciliary beating in three dimensions: steps of a quantitative description

We document a novel approach for quantitative assessment of ciliary activity, exemplified in rapid three-dimensional cyclic motion of the frontal cirri of Stylonychia. Cells held under voltage-clamp control are stimulated by step pulses to elicit reproducible hyperpolarization- or depolarization-induced ciliary motor responses. High-speed video recording at 200 fields per second is used for imaging ciliary organelles of the same cell in two perspectives: the axial view and, following cell rotation by 90 degrees, the lateral view. From video sequences of typically 1 s, the contours of the cirral images are determined and digitized. Computer programs are established to (1) reduce an observed image to a "ciliary axis", (2) sort series of axes by template to generate an averaged ciliary cycle in 2D-projection, and (3) to associate the generalized axial and lateral 2D-images for generation of a sequence of three-dimensional images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined perspectives of images selected from the ciliary cycle, and to generate stereo views for graphical representation of ciliary motion. The approach includes a potential for extraction of the complete microtubular sliding program of a cilium under reproducible electric stimulation of the ciliary membrane.