Chapter 34 High-speed Video Microscopy of Flagella and Cilia

Abstract

Publisher Summary The calculation of beat frequency of ciliary and flagellar movements requires use of a high-speed recording system. A sufficiently large number of images must be recorded over a short enough time to provide detailed information on the waveform of a cilium or flagellum throughout a single beat cycle. This is particularly important when studying transient phenomena, such as chemotactic or phototactic steering. Solutions to this problem include the use of video cameras capable of providing multiple images per field or camera and recorder systems capable of capturing greater than 60 fields per second. The image must be captured over a sufficiently short period that a rapidly moving cilium or flagellum is sharply defined. This chapter briefly describes the advantages and disadvantages of several high-speed video systems that have been used to observe and record ciliary and sperm flagellar movements. A video camera equipped with a rapid shutter will freeze beating cilia and flagella onto videotape at 60 fields per second using a standard videocassette recorder (VCR), with still-field and field-by-field advance capabilities for analysis of images. A video camera with four vertical scanning frequencies of 60, 120, 180, and 240 Hz produces one, two, three, and four separate images per field, yielding up to 240 successive images per second. Such a camera is useful for recording rapidly beating flagella and flagella that are undergoing transient changes in beat pattern. The Nac MHS-200 captures sharp images of beating cilia and flagella using a phase-contrast microscope and the strobe supplied with the system. Stroboscopic illumination is suited to applications where the object being recorded is moving rapidly and must be frozen in time to obtain a sharp image. For high-speed video microscopy of cilia and sperm flagella, a phase-contrast or differential-interference–contrast microscope is suitable. The chapter also discusses the detailed procedure for recording.