Three-dimensional vibration of the malleus and incus in the living gerbil.

In previous studies, 3D motion of the middle-ear ossicles in cat and human was explored, but models for hearing research have shifted in the last few decades to smaller mammals, and gerbil, in particular, has become a popular hearing model. In the present study, we have measured with an optical interferometer the 3D motion of the malleus and incus in anesthetized gerbil for sound of moderate intensity (90-dB sound pressure level) over a broad frequency range. To access the ossicles, the pars flaccida was removed exposing the neck and head of the malleus and the incus from the malleus-incus joint to the plate of the lenticular process. Vibration measurements were done at six to eight points per ossicle while the angle of observation was varied over approximately 30 ° to enable calculation of the 3D rigid-body velocity components. These components were expressed in an intrinsic reference frame, with one axis along the anatomical suspension axis of the malleus-incus block and a second axis along the stapes piston direction. Another way of describing the motion that does not assume an a priori rotation axis is to calculate the instantaneous rotation axis (screw axis) of the malleus/incus motion. Only at frequencies below a few kilohertz did the screw axis have a maximum rotation in a direction close to that of the ligament axis. A slight slippage in the malleus-incus joint developed with increasing frequency. Our findings are useful in determining the sound transfer characteristics through the middle ear and serve as a reference for validation of mathematical middle-ear models. Last but not least, comparing our present results in gerbil with those of previously measured species (human and cat) exposes similarities and dissimilarities among them.

Simultaneous measurements of ossicular velocity and intracochlear pressure leading to the cochlear input impedance in gerbil.

Recent measurements of three-dimensional stapes motion in gerbil indicated that the piston component of stapes motion was the primary contributor to intracochlear pressure. In order to make a detailed correlation between stapes piston motion and intracochlear pressure behind the stapes, simultaneous pressure and motion measurements were undertaken. We found that the scala vestibuli pressure followed the piston component of the stapes velocity with high fidelity, reinforcing our previous finding that the piston motion of the stapes was the main stimulus to the cochlea. The present data allowed us to calculate cochlear input impedance and power flow into the cochlea. Both the amplitude and phase of the impedance were quite flat with frequency from 3 kHz to at least 30 kHz, with a phase that was primarily resistive. With constant stimulus pressure in the ear canal the intracochlear pressure at the stapes has been previously shown to be approximately flat with frequency through a wide range, and coupling that result with the present findings indicates that the power that flows into the cochlea is quite flat from about 3 to 30 kHz. The observed wide-band intracochlear pressure and power flow are consistent with the wide-band audiogram of the gerbil.

Improved correction of axial geometrical distortion in index-mismatched fluorescent confocal microscopic images using high-aperture objective lenses.

Extracting quantitative data from microscopic volume images is straightforward when the refractive indices of the immersion medium and the mounting medium are equal. The readings of the position of the specimen stage can be directly used to measure depth and width. Imperfectly matched immersion and mounting media result in axial geometrical distortion. Linear correction of the axial distortion using the paraxial estimate of the axial scaling factor yields results that may differ as much as 4% from the actual values. From calculations based on a theoretical expression of the 3-D point-spread function in the focal region of a high-aperture microscope focussing into a mismatched mounting medium, we derived axial scaling factors that result in quantitative results accurate to better than 1%. From a non-linear correction procedure, an improved formula for the paraxial estimate of the axial scaling factor is derived.

Scala vestibuli pressure and three-dimensional stapes velocity measured in direct succession in gerbil.

It was shown that the mode of vibration of the stapes has a predominant piston component but rotations producing tilt of the footplate are also present. Tilt and piston components vary with frequency. Separately it was shown that the pressure gain between ear canal and scala vestibuli was a remarkably flat and smooth function of frequency. Is tilt functional contributing to the pressure in the scala vestibuli and helping in smoothing the pressure gain? In experiments on gerbil the pressure in the scala vestibuli directly behind the footplate was measured while recording simultaneously the pressure produced by the sound source in the ear canal. Successively the three-dimensional motion of the stapes was measured in the same animal. Combining the vibration measurements with an anatomical shape measurement from a micro-CT (CT: computed tomography) scan the piston-like motion and the tilt of the footplate was calculated and correlated to the corresponding scala vestibuli pressure curves. No evidence was found for the hypothesis that dips in the piston velocity are filled by peaks in tilt in a systematic way to produce a smooth middle ear pressure gain function. The present data allowed calculations of the individual cochlear input impedances.

Thickness distribution of fresh eardrums of cat obtained with confocal microscopy.

The aim of this study was to measure the spatial thickness distribution of the cat tympanic membrane (TM), a very thin, virtually transparent and delicate biological membrane. Axial fluorescence images taken perpendicular through isolated TM were recorded for five different cats using confocal laser scanning microscopy. Thickness was measured on the cross-section of the membranes in the axial images. A correction for focal shift due to refractive-index mismatch was applied. Similar thickness distributions were obtained in all measured samples (n = 9). The pars tensa had a rather constant thickness in the central region between the annulus and manubrium. The thickness increased steeply toward the peripheral rim. Thickness was smallest in the inferior region, with values ranging between 5.5 and 9 microm in the central part and up to 50 microm near the annulus. More superiorly, thickness was slightly higher, up to 20 microm, between the annulus and manubrium. The anterior part was thicker than the posterior side. These findings are strongly different from a current value in the literature. Our data allow a more precise representation of the eardrum in mathematical models, which are a prerequisite for a better understanding of middle-ear mechanics. The optical sectioning technique of the confocal microscope did not result in any preparation artifacts and was therefore also used to quantify shrinkage due to preparation of histological sections of TMs.

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch.

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 microm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water-polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.

A simple method for checking the illumination profile in a laser scanning microscope and the dependence of resolution on this profile.

One of the conditions for a laser scanning microscope to reach its optimal performance is for it to operate at its full numerical aperture (NA). In most commonly used systems, the illumination intensity at the back focal plane of the objective lens is apodized. This paper presents a simple method using a photodiode for checking the actual illumination intensity profile. We show as an example the measured profiles of a laser beam when working with two high-NA immersion objectives in two different confocal systems, and also show that in theoretical studies of the point-spread function, the assumption of a flat compared with a truncated Gaussian beam profile gives rise to severe discrepancies. The measured profiles also serve as an indication of the necessity of a realignment of the optical system.

Three-dimensional modelling of the middle-ear ossicular chain using a commercial high-resolution X-ray CT scanner.

The quantitative measurement of the three-dimensional (3-D) anatomy of the ear is of great importance in the making of teaching models and the design of mathematical models of parts of the ear, and also for the interpretation and presentation of experimental results. This article describes how we used virtual sections from a commercial high-resolution X-ray computed tomography (CT) scanner to make realistic 3-D anatomical models for various applications in our middle-ear research. The important problem of registration of the 3-D model within the experimental reference frame is discussed. The commercial X-ray CT apparatus is also compared with X-ray CT using synchrotron radiation, with magnetic resonance microscopy, with fluorescence optical sectioning, and with physical (histological) serial sections.

Effect of middle ear components on eardrum quasi-static deformation.

Eardrum deformation induced by quasi-static middle ear pressure was studied at progressive stages of dissection of gerbil temporal bones. With our high resolution moiré interferometer we recorded the shape and deformation of the eardrum along a line perpendicular to the manubrium and through the umbo, at different middle ear pressures. The deformation was measured from the medial side, after serially removing the cochlea, removing the stapes, cutting the tensor tympani, exposing the incudo-mallear joint, and cutting the anterior bony process which connects the malleus to the tympanic bone. The mean displacement as a function of pressure was also determined at all stages of dissection. Removing the cochlea and stapes, and cutting tensor tympani has no effect on static eardrum deformation. Exposing the incudo-mallear joint increases eardrum movement, and cutting the anterior bony connection between malleus and temporal bone strongly changes eardrum rest position and further increases its displacement.

Displacement pattern of the normal pars flaccida in the gerbil.

OBJECTIVE: The aim of the current study was to assess the mechanical stiffness properties of the normal pars flaccida and to compare the results with those obtained in earlier studies on the pars tensa. BACKGROUND: Postinflammatory changes such as retraction pockets and cholesteatoma develop in the pars flaccida as well as in the pars tensa of the tympanic membrane. In these authors' previous experimental studies, stiffness changes are shown to develop early in the pars tensa in response to purulent otitis media and otitis media with effusion. These changes are suggested to be precursors to a later development of retraction pockets and cholesteatoma. In the clinical situation, retraction pockets are often found in the pars flaccida only. This study will establish the stiffness properties of the normal pars flaccida and form a base for forthcoming studies of the pars flaccida in response to otitis media with effusion and purulent otitis media, as well as retraction pocket formation and cholesteatoma. METHODS: A measure for the mechanical stiffness properties of the normal pars flaccida in the gerbil was assessed as its displacement for a given transtympanic pressure. The method used was moiré interferometry, which is a noncontacting optical technique to measure the shape of the surface of an object. RESULTS: The displacement of the pars flaccida was a nonlinear and asymmetric function of pressure. The displacement per pressure unit rose steeply at low middle ear pressures to level out and reach a steady state at higher pressures. The displacement versus pressure characteristics for the pars flaccida strongly differed from those of the pars tensa. The pars tensa seemed more elastic. CONCLUSION: Reference values for displacement versus pressure characteristics of the normal gerbil pars flaccida were obtained using a moiré interferometry method. The mechanical stiffness properties of the normal pars flaccida were strongly different from those of the pars tensa.

Systematic errors in small deformations measured by use of shadow-moiré topography.

Phase-shift shadow-moiré topography is a noncontact optical technique for measuring the shapes of surfaces. Artifactual bands resembling isoheight surface contours are observed during measurement of small changes in shape by use of this technique. The shape-reconstruction algorithm used in shadow-moiré topography is based on a mathematical model of the fringe patterns generated on the surface to be measured. We hypothesize that the observed bands reflect systematic errors caused by ignoring height-dependent terms in the mathematical model of the fringe patterns. We test the assumption by simulating the fringe patterns for a virtual test surface by using a model that contains height-dependent terms and one term that is idealized by ignoring these terms. Small systematic errors in shape are observed only when the surface is reconstructed from fringe patterns simulated with a model containing the height-dependent terms. Shape-error curves are computed as a function of the surface height by the subtraction of the reconstructed shape from the known shape. Simulated shape-error curves agree with experimental measurements in that they show an increase in error with surface height, and both the experimental and the simulated shape-error curves contain ripples. Although the errors are small in comparison with the dimensions of the surface and are negligible in shape measurements and in most deformation measurements, they may show up as noticeable bands in images of small deformations.

Tympanic membrane displacement patterns in experimental cholesteatoma.

Tympanic membrane (TM) stiffness changes in the pars tensa in response to experimentally induced ear canal cholesteatoma by obstruction of the ear canal were studied. To this aim TM displacement versus pressure was measured with a high resolution, differential moiré interferometer. The measurements were performed on fresh, isolated gerbil temporal bones after removal of the cholesteatoma bulk. Besides an overall stiffness reduction we found that local stiffness variations were present in nine out of 18 studied ears. The stiffness changes as a function of time after ear canal obstruction had a pattern similar to those previously shown to develop in response to various forms of otitis media, showing that the TM stiffness properties decrease in a similar way in response to different inflammatory middle ear diseases. The stiffness changes correlated with an increased overall TM thickness and increased thickness of the lamina propria in particular as measured in histology sections. The stiffness changes may play an important role in the pathophysiology of cholesteatoma.

Volume displacement of the gerbil eardrum pars flaccida as a function of middle ear pressure.

The pars flaccida (PF) is a small region of the eardrum, with elasticity and histology completely different from the rest of the membrane, which has often been attributed a pressure regulating function for the middle ear (ME). In this paper, the volume displacement of the PF as a function of ME pressure is discussed. The deformation of the PF was measured in vitro in five Mongolian gerbil ears, by means of an opto-electronic moiré interferometer. Volume displacement was determined at small intervals in three sequential pressure cycles, in the range of +/- 0.4 kPa, +/- 2 kPa, and again +/- 0.4 kPa. The displacement was found to be a highly non-linear function of pressure, with a strong increase up to 0.4 kPa ME over- or underpressure and remaining nearly unchanged for pressures beyond 0.4 kPa. In all animals, maximal volume displacement was less than 0.5 microl, or 0.2% of total ME air volume. Clear hysteresis was found between the deformations at the same pressure level in the increasing and decreasing parts of the pressure cycles. Membrane behavior in the first 0.4 kPa pressure cycle was significantly different from that in the second 0.4 kPa cycle, which followed the 2 kPa pressure cycle. The results indicate that the ME pressure change regulation function of the PF is limited to very small pressure changes of a few hundred Pa around ambient pressure, and that larger ME pressures cause at least short-term changes in the membrane's behavior.

Measurement and modeling of boundary shape and surface deformation of the Mongolian gerbil pars flaccida.

The shape of the pars flaccida (PF) boundary and its pressure induced deformation was measured in five Mongolian gerbil ears, using an opto-electronic moiré interferometer. To determine the PF boundary, membranes were deformed by middle ear (ME) pressures of -2 kPa and +2 kPa. The boundary of the PF was defined as the locus of points where the pressure induced deformation begins. To a very high precision, this boundary was found to be a circle with the same radius for both over- and underpressure deformations. Between animals the radius varied from 0.760 mm to 0.778 mm. We show that the shape of the PF, while being deformed by static pressure in the ME, can be modeled as part of a sphere. Volume displacement can then be calculated as the volume enclosed by the sphere cap, delimited by the circular PF boundary plane. Volume displacement was calculated for membranes deformed by 400 Pa ME overpressure and -400 Pa ME underpressure. The agreement of the experimental data with the sphere cap model is shown to be very good, and results in a volume displacement measuring accuracy which is better than 10%.

Coating techniques in optical interferometric metrology.

In optical interferometry diffuse reflectivity of the surface under study should be high and homogeneous. Application of a white reflective coating can strongly improve measurement results. The optical properties of bronze powder, TiO(2) powder, white Chinese ink, and MgO coatings are discussed. Measurements of reflected intensity distribution show that white Chinese ink and MgO have superior optical characteristics, and electron microscopy shows that these coatings cause thickness artifacts of less than 7.5 and 17 mum, respectively. The effect on deformation measurements is demonstrated by moiré topography on a thin membrane that is deformed under small static pressures. The membrane center displacement varies from 15 to 100 mum, and within a measuring precision of 2.5 mum no artifacts on this deformation are found.

Tympanic membrane changes in experimental purulent otitis media.

The stiffness properties of the gerbil tympanic membrane (TM) were investigated during the early course of experimental purulent otitis media produced by middle ear inoculation (n = 49) with type 3 Streptococcus pneumoniae. In a first approach the acoustic admittance and susceptance were measured in vitro with tympanometry and in a second approach the tympanic membrane displacement in response to static pressure was measured with moire interferometry. A histological assessment of the tympanic membrane was made concurrently. The acoustic admittance and susceptance reduced during the initial days post-inoculation. These reductions seem to correlate with an oedema simultaneously developing in the pars tensa. The pressure-induced displacement increased with time of disease, i.e. the mechanical stiffness of the TM reduced. Local deformation zones, or 'weak spots' appeared in the inferior half of the pars tensa in three out of eight cases measured at 3 or 4 days post-inoculation. The loss of stiffness in the pars tensa may affect the further course of otitis media, and the 'weak spots' are possible precursors of retraction pockets and/or perforations. The stiffness reductions may be caused by changes on a sub-microscopical, molecular level.

Acoustically induced vibrations of the Reissner's membrane in the guinea-pig inner ear.

In the inner ear, the Reissner's membrane separates the scala vestibuli from the scala media and is thus of importance for maintaining a positive endocochlear potential. The motion of the membrane is thought to be driven by the vibrations of the underlying hearing organ caused by a hydromechanical coupling between the structures. Since the Reissner's membrane is relatively easily accessible in the cochlea its vibratory response has been used as a measure of the micromechanical behaviour of the hearing organ. To determine whether this indirect measure revealed the true characteristics of the hearing organ, experiments were performed using laser heterodyne interferometry in an in vitro preparation of the guinea-pig temporal bone. Interferometric measurements at the Reissner's membrane and at the surface of the hearing organ directly beneath made it possible to compare the mechanical tuning characteristics of both structures. It was found that the mechanical response characteristics of the Reissner's membrane differed considerably from the hearing organ. The tuning frequency was different and only minor changes in the maximal vibration amplitude were seen when measuring at different radial locations. However, the shape of the response curve changes with location. The Reissner's membrane response appeared to be affected by the mechanical vibrations originating both at the middle ear ossicles and at the hearing organ. It is concluded that the Reissner's membrane response is a poor indicator of cochlear mechanics and that investigations of cochlear micromechanics should be performed directly at the level of the hearing organ.

On the incorporation of moiré shape measurements in finite-element models of the cat eardrum.

The mechanical behavior of the eardrum has previously been shown to depend critically on its shape, but accurate shape measurements have been difficult to make. Phase-shift moiré topography provides a valuable technique for measuring such shapes, and measurement in the presence of large static pressures facilitates the determination of the boundaries of the pars tensa, pars flaccida, and manubrium. New measurements of the shape of the cat eardrum are presented. The presence of hysteresis in the pressure-displacement response is demonstrated. The shapes are incorporated in individualized finite-element models for four different ears, and the variability between and within animals is examined. Fixed-manubrium low-frequency displacements are simulated and compared for the different models.