© 2006 The Authors Journal compilation © 2006 The Royal Microscopical Society Journal of Microscopy, Vol. 223, Pt 2 August 2006, pp. 159–164 Received 13 December 2005; accepted 10 March 2006 Blackwell Publishing Ltd SHORT COMMUNICATION Use of confocal linescan to document ciliary beat frequency R. T. DOYLE, T. MONINGER*, N. DEBAVALYA† & W. H. HSU† Department of Genetics, Development and Cell Biology, Roy J. Carver Laboratory of Ultrahigh Resolution Biological Microscopy, Institute for Combinatorial Discovery, Iowa State University, Ames, IA 50011, U.S.A. *Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. †Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, U.S.A. Key words. Brachionus quadridentatus, ciliary beat frequency, ciliated trachael cells, confocal linescan, linescan analysis. Received 13 December 2005 ; accepted 10 March 2006 Summary We present a method to document ciliary beat frequency with the linescan function of a scanning confocal microscope, using ciliated tracheal cells and free-swimming rotifers as examples. Depending on the clarity of the original data, the ciliary beat frequency can be determined from the confocal linescan directly or from an intensity linescan analysis of the original data. Fast Fourier transform treatment of the data can be used to verify the derived ciliary beat frequency. The linescan approach allows analysis of simple ciliary movements dis- played by the ciliated tracheal cells, as well as complex move- ments performed by free-swimming rotifers while feeding. Introduction The determination of the ciliary beat frequency (CBF) of cili- ated cells has been performed for many years. Collection of these data has always been closely associated either with tech- niques that allow rapid collection of images of the beating cilia (Salathe & Bookman, 1999; Li et al., 2000; Nlend et al., 2002; Hirst et al., 2003, 2004; Sisson et al., 2003; Wyatt et al., 2003; Zhang & Sanderson, 2003; Piatti et al., 2004; Robertson et al., 2004; Shiima-Kinoshita et al., 2004) or with optical methods to document the light intensity changes imparted by beating cilia (Dalhamn & Rylander, 1962; Naito & Kaneko, 1973; Mercke et al ., 1974; Lee & Verdugo, 1976, 1977; Verdugo, 1980; Puchelle et al., 1982; Verdugo & Golborne, 1988; Devalia et al., 1990; Bayram et al., 1998; Thanou et al., 1999; Dimova et al., 2003; Barrera et al., 2004). Image-based methods result in long and painstaking analysis of frame-by-frame observation of cilial movement to document the CBF. Photocell- and photomultiplier-based methods produce real-time light intensity records that can be saved and analysed or processed on the fly to produce fast Fourier trans- form (FFT) interpretation of the frequency records; this helps to position and focus the optical system in real time to optimize data collection (Salathe & Bookman, 1999; Nlend et al., 2002). Here we report a photomultiplier-based technique to deter- mine CBF using the linescan function of a scanning confocal microscope. Both cultured porcine ciliated tracheal cells and free-living Brachionus rotifers were imaged to test this system. We used the 512-linescan function of a Prairie Technologies (Middleton, WI, U.S.A.) scanning confocal microscope to pro- duce a light intensity record of the active ciliated cell, and then produced a light intensity data record with the linescan analysis function of the image analysis software metamorph (Universal Imaging Corporation, Downingtown, PA). Depending on the clarity of the cilia frequency record, the data can be analysed by counting the intensity cycles in the confocal linescan record or the metamorph record and converting that to CBF. The data can also be imported into software for FFT analysis. Materials and methods Preparation of cultured porcine ciliated tracheal cells Epithelial cells were isolated from healthy 3–6 month old specific-pathogen-free pigs. The methods of cell isolation were modified from those of Young et al. (2000). Mid-portions of tracheas were aseptically collected in cold phosphate buffered saline and were transported to the laboratory. Upon arrival, the connective tissues were aseptically removed from the tracheas, leaving only the membranous and cartilaginous parts. The tracheas were each rinsed and transferred into separated centrifuge tubes. Tracheal epithelial cells were dissociated by chilled enzyme solution containing 0.1% DNase and 0.005% pronase in Ca 2+ - and Mg 2+ -free minimum essential medium (5.37 mm KCl, 110 mm NaCl, 44 mm NaHCO 3 , 0.91 mm Correspondence to: R. T. Doyle. Tel: 515 294 6513; fax: 515 294 7134; e-mail: rtdoyle@iastate.edu