Laser interferometric flow measurements in the lateral line organ Tsang, P

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1 University of Groningen Laser interferometric flow measurements in the lateral line organ Tsang, P IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1997 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Tsang, P. (1997). Laser interferometric flow measurements in the lateral line organ Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 1 maximum. Download date:

2 3 Fluid flow profiles measured in the supraorbital lateral line canal of the ruffe Introduction Fishes detect water motion by means of a lateral line organ. One type of lateral line organ is located in recessed canals on the head of the fish. Water motion outside the fish is passed on via skin membranes (van Netten and van Maarseveen, 1994) to canal fluid which in turn drives a number of dome shaped structures, called cupulae. The cupular motion is sensed by stereocilia of hair cells attached to the base of the cupula (Kroese and van Netten, 1989). The lateral line organ is easily accessible and thus offers the opportunity for the study of a sensitive hair cell organ in a relatively undisturbed natural condition. Cupular motion in response to an oscillating stimulus sphere placed in the canal has been measured (van Netten and Kroese, 1987), but little data exist regarding the fluid flow within the canal. So far, the analyses of lateral line mechanics have neglected the influence of the canal wall assuming the flow driving the cupula to be spatially uniform (van Netten, 1991). Measurements of fluid motion in the lateral line canal of sprats using seeding particles have been conducted (Denton and Gray, 1983), but these were limited to a single point within the canal. Free floating seeding particles suffer from Brownian motion and convection, making them unsuitable for monitoring canal fluid flow at physiological stimulus levels. This led to the development of a sense probe that follows the fluid motion, with a measuring volume fixed in place and sensitive enough to detect flow velocities down to the order of 1 µm/s. To further our understanding of the stimulus transduction in the peripheral lateral line organ, the influence of the canal wall on the fluid stimulus was studied. Methods The ruffes, Acerina cernua (L), used in these experiments were anaesthetised by I.P. injection of Saffan (25 mg/kg) (Pitman Moore) (Oswald, 1978) and placed in a small tank where they were fixed in position by head and body clamps. 27

3 f = 1 mm l /2 waveplate Bragg-cells 8 MHz 79.6 MHz f=8mm attenuator imaging system f= mm l/4 waveplate l/2 waveplate photodetector laser beams stimulus sense probe Ruffe ( Acerina cernua L.) bony bridge lateral line canal Figure 1: The laser interferometer used for the velocity measurements is very similar to the one described by van Netten (van Netten, 1988). The two laser beams emerge from the Bragg-cells with an optical frequency difference of 4 khz between them. They are directed via a series of mirrors through a polarising beamsplitter and l/4 waveplate before finally being brought to focus upon the surface of the sense probe. The sense probe, driven by the fluid flow, Doppler-shifts the laser light and some of the scattered light travels back along the optical axis and is focused onto the photodetector. An image of the sense probe can be redirected to the imaging system by adjusting the l/2 waveplate. The cephalic lateral line canals consist of skin covered cartilaginous canals, approximately 1 mm deep and 1.8 mm wide, with bony bridges covering the 28

4 cupulae. For the experiments, a small section of the skin above the supraorbital canal was removed to expose cupula II (following the notation of Jakubowski (Jakubowski, 1963) and the region just behind cupula I. The experiments described here were performed in empty lateral line canals from which the epithelium and cupula II were removed, while the bony bridge above cupula II was left intact. To measure the fluid flow at a selected position within the canal, a sense probe consisting of a small sphere (Æ 5 mm) attached to a highly flexible tapered borosilicate glass fibre was used. The glass fibre is around 2.5 mm long and has a diameter of about 4 mm (see Fig. 1). The sense probe is mounted on a x,y,zpositioner and can be positioned at any location in the fluid. The positions of the canal walls and the bony bridge were determined by scanning the laser beams from one side to the other. The sense probe was then positioned in the midpoint of the canal at approximately.3 mm caudal from the bony bridge. A small stimulus sphere (Æ.67 mm) driven by a piezoelectric stimulator was then placed on the other side of the bony bridge just behind cupula I in order to produce a sinusoidally changing fluid flow in the canal. The magnitude of the stimulus sphere s velocity was kept at 3.14 mm/s over the entire frequency range at which we measured. The velocity of the sense probe moving in response to the evoked fluid motion was measured with a heterodyne laser interferometer at different depths in the canal. The sense probe was calibrated by measuring its response to a large (Æ 4 mm) stimulus ball vibrating sinusoidally in free field, placed a few mm away from it. The response was found to be frequency independent as expected from theory (van Netten, 1991). In addition, these calibration measurements confirmed that, at the frequencies used, the sense probe s motion is equal to that of the calculated fluid motion to within a few percent (see Fig. 2). The fish was artificially respired with tap water throughout the duration of the experiment. The flow of water was stopped during the measurements to avoid unwanted vibrations caused by the flow of water through the gills. The condition of the fish was checked periodically by looking at the blood flowing through a blood vessel inside the eye. The temperature of the surroundings (15 C) was kept constant throughout the duration of the experiment. This slowed the formation of air bubbles in the fish tank and reduced the production of fish slime in the canal. 29

5 1 amplitude (µm/s) measurements theory distance from the edge of the stimulus ball (mm) Figure 2: A plot of the free field water flow velocity as a function of horizontal distance away from the edge of the stimulus ball, as measured with the sense probe. The stimulus ball has a radius of 2 mm and is vibrating at 1 Hz with an amplitude of 6.75 µm. The solid line is the velocity calculated from theory (van Netten, 1991) and shows that the sense probe's motion is reliably following the fluid flow. Results First, measurements were conducted in free field at the same positions with regard to the stimulus sphere and sense probe as to be used in the canal experiments, to isolate the mechanical effects of the lateral line canal. The amplitude and phase of the fluid flow velocity in free field are shown in Fig. 3, they have a fairly flat profile for all frequencies, as expected from calculations on the fluid field caused by a vibrating sphere in a large fluid volume (van Netten, 1991) for the geometry used. 3

6 amplitude (µm/s) vertical distance (µm) 1 Hz 3 Hz 7 Hz phase (deg) vertical distance (µm) Figure 3: A plot of the amplitude and phase of the velocity as a function of vertical distance from the bottom edge of the stimulus sphere as measured with the sense probe in free field at 2.5 mm away from the stimulus sphere for different stimulus frequencies. The data from, 4, 6 Hz were not plotted, since they do not significantly differ from the data plotted above. These characteristics are to be compared with Fig. 4, which show typical measurements in the lateral line canal at various depths and frequencies. For all the fishes investigated, the flow pattern measured in the empty lateral line canal is dependent on the frequency of the stimulus. The smallest separation between the sense probe and the canal floor is approximately 8 mm (allowing room for movement) and together with the radius of the sense probe (25 mm), it means that measurements started at a distance of 33 mm. Clearly, the profiles found in the lateral line canal differ significantly from those measured in free field (Fig. 3), demonstrating that the canal plays an important role in shaping the actual stimulus to the cupula. amplitude ( µm/s) distance from the canal floor (µm) phase (deg) Hz Hz 3 Hz 4 Hz 6 Hz 7 Hz distance from the canal floor (µm) Figure 4: A plot of the amplitude and phase of the velocities measured at different depths in the empty canal for a range of stimulus frequencies. The distance to the edge of the bony bridge is.3 mm. 31

7 The maximum of the profile in the canal at 1 Hz is found at approximately 4 mm above the canal floor. As the stimulus frequency increases, the maximum gradually shifts to a value of about 15 mm at the higher frequencies used. For still higher frequencies (results not shown), the profiles do not change significantly from this. The phase shown in Fig. 4 is very different from that of Fig. 3. There is a phase lead near the canal wall, but as the distance away from the floor is increased it changes to a phase lag. From 6 mm onwards the phase is heading towards zero, as the edge of the canal is approached (nearing free field). This was found for all the frequencies at which we measured. An increase in stimulus frequency has the effect of flattening the phase found at the mid region of the canal. Discussion The experimental results clearly indicate the influence of the lateral line canal's boundary layer. In contrast to measurements made in free field (Fig. 3), the profiles measured in the lateral line canal (Fig. 4) not only show a dependence on vertical distance but also depend on stimulus frequency. To further characterise the effect of the lateral line canal wall on the fluid flow, the experimental results can be compared with two extreme models that may simulate the canal wall. One model consists of fluid flowing past an (infinite) plate, while the other model considered is fluid flow inside an (infinitely) long tube with radius r. Clearly, the behaviour of lateral line canal fluid is expected to behave in an intermediate fashion, since the lateral line canal consists of grooves recessed in bone and is, at least in a hydrodynamic sense, partially open because of the existence of the skin covered openings in the bone. Moreover, during measurements the skin covering these openings was removed. The fluid velocities in both models can be solved in analytical terms (Schlichting, 1987) for (spatially) constant pressure gradients, and both can be characterised by an a.c. boundary layer thickness defined by d=(2µ/ωρ) 1/2, which gives a measure of the distance over which the fluid is significantly affected by either the plate or the tube s wall. Here, m is the fluid viscosity (1 mpa s for water), w is the stimulus frequency, and r is the fluid density (1 kg/m 3 ). This yields for δ approximately 18 mm at 1 Hz and 7 mm at 7 Hz. The a.c. fluid flow past a plate increases monotonically from zero at the plate s surface, to the free field value far from the plate. This transition occurs within a characteristic distance equal to d. This behaviour is unlike the experimental results. 32

8 The profiles measured show maxima over the canal cross-section, similar to the behaviour of fluid in tubes. The measured shift of the maximum towards the canal floor, as frequency increases, follows the pattern in a long tube in which at low frequencies the profile is parabolic with a maximum in the centre. At high frequencies the profile has a ring-like shape with maxima not in the centre but at a frequency dependent distance from the wall given by (Schlichting, 1987; Sexl, 193; Womersley, 1955): D max = 2.28 d. Substituting the high stimulus frequency used (7 Hz) results in 15 µm, which is comparable to the position of the maxima found in the lateral line canal at this frequency. Thus, although the canal was open during the measurements, the profiles measured compare favourably with that of a long closed tube. Conclusions The novel method we employ for in vivo flow measurement is sensitive enough to detect velocities down to 1 µm/s at a spatial resolution of 5 µm. The flow profiles obtained for an empty lateral line canal clearly demonstrate that the cupula receives a frequency dependent stimulus, which deviates from a flat profile. The fluid flow in the canal is comparable to that in a long tube. The stimulus to the cupula, therefore, is controlled by the boundary layer of the canal. Experiments are now underway to investigate the flow profile and boundary layer generated by the integral structure of canal wall and cupula. An interesting consequence of the position of the profile maxima found in the lateral line canal may be that to detect frequencies below 1 Hz, the canal cupulae should extend to at least 15 µm from the canal floor into the fluid to benefit from the maxima in flow velocity. This condition is certainly met in the case of the ruffe where the cupulae have an average height of approximately 6 µm. Acknowledgements We are very grateful to Prof. H. Duifhuis, P. W. J van Hengel, C. J. Kros, M. P. M. G. van den Raadt and J. E. C Wiersinga-Post for their comments on an earlier version of this manuscript. The investigations were supported by the Life Sciences Foundation (SLW) which is subsidized by the Netherlands Organisation for Scientific Research (NWO). 33

9 References Denton, E. J. and Gray, J. A. B. (1983). Mechanical factors in the excitation of clupeid lateral lines. Proc. R. Soc. Lond. Biol. Sci., 218: Jakubowski, M. (1963). Cutaneous sense organs of fishes. I. The lateral-line organs in the stone-perch (Acerina cernua L.), Acta Biol. Cracoveniensia, Zool. 6, Kroese, A. B. A. and Netten, S. M. van (1989). Hair cell transduction. In: The mechanosensory lateral line (Neurobiology and evolution) (ed. by S. Coombs, P. Görner and H. Münz), pp Springer-Verlag. Netten, S. M. van and Kroese, A. B. A. (1987). Laser interferometric measurements on the dynamic behaviour of the cupula in the fish lateral line. Hearing Res. 29, Netten, S. M. van (1988). Laser interferometer microscope for the measurement of nanometer vibrational displacements of a light-scattering microscopic object. J. Acoust. Soc. Am. 83, Netten, S. M. van (1991). Hydrodynamics of the excitation of the cupula in the fish canal lateral line. J. Acoust. Soc. Am. 89, Netten, S. M. van and Maarseveen, J. Th. P. W. van (1994). Mechanophysiological properties of the supraorbital lateral line canal in ruffe (Acerina cernua L.) Proc. R. Soc. Lond. B 256, Oswald, R. L. (1978). Injection anaesthesia for experimental studies in fish. Comp. Biochem. Physiol. 6C, Schlichting, H. (1987). Boundary-layer theory (McGraw-Hill Publishing Company, New York). Sexl, T. (193) Über den von E.G. Richardson entdeckten "Annulareffekt". Z.Phys. 61, Womersley, J. R., (1955). Method for the calculation of velocity, rate of flow and viscous drag in arteries when the pressure gradient is known, J. Physiol. 127,