Chapter 41 Quantification of Ciliary Beat Frequency and Metachrony by High-Speed Digital Video

doi:10.1016/S0091-679X(08)60822-5

Methods in Cell Biology

Volume 47, 1995, Pages 289-297

https://doi.org/10.1016/S0091-679X(08)60822-5 Get rights and content

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The quantification of ciliary activity depends on the detection and analysis of the rhythmic variation in light intensity of a microscope image induced by ciliary movement. This approach requires the simplification and optimization of signal contrast and waveform and its success depends on the photo-detection method, the microscope optics, and the type of cell preparation used. For beat frequency measurements, a single detector system consisting of a photomultiplier, phototransistor, or fiberoptic device is sufficient. For the analysis of ciliary metachrony, spatial information is essential and a system requires a multipoint detector such as a digital video camera. This chapter highlights the advantages and limitations of earlier techniques for measuring ciliary activity and describes the principles and methodology of an advanced high-speed digital video technique. Large light signal amplitude can be obtained from coordinated groups of active cilia of isolated or cultured cells with phase-contrast optics. The signal waveform needs to be simple and easily interpretable; however, cilia of isolated cells or epithelial sheets generally beat in a metachronal manner and this can result in a complicated signal waveform because of the orientation of the ciliary beat cycle with respect to the detector and the number of cilia simultaneously contributing to the signal. An improvement in signal waveform can be achieved by monitoring only the effective stroke by recording close to the ciliary tips. The chapter discusses quantification of ciliary metachronism and video techniques for measuring ciliary activity. The high-speed digital video photodetection technique discussed has a high temporal and spatial resolution that can be used for the measurement of ciliary beat frequency and metachrony. The combination of this technique with a fluorescence imaging system that can simultaneously measure intracellular ions or messengers provides a unique opportunity for investigation of the physiological control of ciliary activity.

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Cited by (12)

Respiratory Ciliary Dysfunction
2008, Pediatric Respiratory Medicine
High-speed digital microscopy
2000, Methods
High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s⁻¹ (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s⁻¹ (fast), to measure intracellular calcium concentration ([Ca²⁺]_i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca²⁺]_i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca²⁺]_i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.
Regulation of airway ciliary activity by Ca<sup>2+</sup>: Simultaneous measurement of beat frequency and intracellular Ca<sup>2+</sup>
1999, Biophysical Journal
Airway ciliary activity is influenced by [Ca²⁺]_i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca²⁺]_i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s⁻¹) and correlated with [Ca²⁺]_i determined, at the ciliary base, by fast imaging (30 images s⁻¹) of fura-2 fluorescence. As a mechanically induced intercellular Ca²⁺ wave propagated through adjacent cells, [Ca²⁺]_i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca²⁺ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20–23°C, and from 17.2 to 26.7 Hz at 37°C. Changes in [Ca²⁺]_i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca²⁺]_i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca²⁺]_i of ∼250–300 nM.
Nasal mucociliary clearance as a factor in nasal drug delivery
1998, Advanced Drug Delivery Reviews
The nasal mucociliary clearance system transports the mucus layer that covers the nasal epithelium towards the nasopharynx by ciliary beating. Its function is to protect the respiratory system from damage by inhaled substances. Impairment of nasal mucociliary clearance can result in diseases of the upper airways. Therefore, it is important to study the effects of drugs and drug excipients on nasal mucociliary clearance. A large number of methods are used to assess mucociliary clearance. These methods study the effects of drug and excipients on the mucociliary system in vitro or in vivo in animals and humans. In some cases, the results of different in vitro and in vivo measurements do not correlate well. In vitro methods, especially ciliary beat frequency measurements, have been demonstrated to be valuable tools for toxicity screening. However, in vivo studies are essential to confirm the safety of nasal drug formulations. Nasal mucociliary clearance also has implications for nasal drug absorption. Drugs are cleared rapidly from the nasal cavity after intranasal administration, resulting in fast systemic drug absorption. Several approaches are discussed to increase the residence time of drug formulations in the nasal cavity, resulting in improved nasal drug absorption. However, more experimental evidence is needed to support the conclusion that this improved absorption is caused by a longer residence time of the nasal drug formulation.
Directional disorder of ciliary metachronal waves using two-dimensional correlation map
2002, IEEE Transactions on Biomedical Engineering
Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model
2021, Frontiers in Cellular and Infection Microbiology

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Chapter 41 Quantification of Ciliary Beat Frequency and Metachrony by High-Speed Digital Video

Publisher Summary

Biophys. J.

Biol. Cell

Ciliary motion features from digitized video photography.

J. Comput. Assist. Microsc.

In vitro observation and counting methods for ciliary motion

In vivo observation and counting methods for ciliary motion

Spectral characterization of ciliary beating: variations of frequency with time.

Am. J. Physiol.

Characterization of metachronal wave of beating cilia on frog's palate epithelium in tissue culture

J. Physiol. (Lond.)

Metachronal activity of cultured mucociliary epithelium under normal and stimulated conditions.

Cell Motil. Cytoskel.

Automated measurement of ciliary beat frequency.

J. Appl. Physiol.

Analysis of ciliary beating frequency by video-image processing.

J. Comput. Assist. Microsc.