Different rates of endocytic activity and vesicle transport from the apical and synaptic poles of the outer hair cell.

BACKGROUND: Intense endocytic activity at the apex of outer hair cells (OHCs)-the electromechanical cells of the cochlea-has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown. OBJECTIVE: The current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells. MATERIALS AND METHODS: The fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC6 (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles. RESULTS: The plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected. CONCLUSION: The larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.

Objective audiometry with DPOAEs : New findings for generation mechanisms and clinical applications.

BACKGROUND: Distortion product otoacoustic emissions (DPOAEs) and transient-evoked otoacoustic emissions (TEOAEs) are sound waves generated as byproducts of the cochlear amplifier. These are measurable in the auditory canal and represent an objective method for diagnosing functional disorders of the inner ear. Conventional DPOAE and TEOAE methods permit detection of hearing impairment, but with less than desirable accuracy. OBJECTIVE: By accounting for DPOAE generation mechanisms, the aim is to improve the accuracy of inner-ear diagnosis. METHODS: DPOAEs consist of two components, which emerge at different positions along the cochlea and which may cause artifacts due to mutual interference. Here, the two components are separated in the time domain using short stimulus pulses. Optimized stimulus levels facilitate the acquisition of DPOAEs with maximum amplitudes. DPOAE and Békésy audiograms were recorded from 41 subjects in a clinically relevant frequency range of 1.5-6 kHz. RESULTS: The short stimulus pulses allowed artifact-free measurement of DPOAEs. Semilogarithmic input-output functions yielded estimated distortion product thresholds, which were significantly correlated with the subjectively acquired Békésy thresholds. In addition, they allowed detection of hearing impairment from 20 dB HL, with 95% sensitivity and only a 5% false-positive rate. This accuracy was achieved with a measurement time of about 1-2 min per frequency. CONCLUSION: Compared to conventional DPOAE and TEOAE methods, separation of DPOAE components using short-pulse DPOAEs in combination with optimized stimulus parameters considerably enhances the accuracy of DPOAEs for diagnosing impairment of the cochlear amplifier.

[Objective audiometry with DPOAEs : New findings for generation mechanisms and clinical applications. German version]. / Objektive Hördiagnostik mit DPOAE : Neue Erkenntnisse zur Generierung und klinischen Anwendung.

BACKGROUND: Distortion product otoacoustic emissions (DPOAEs) and transient evoked otoacoustic emissions (TEOAEs) are sound waves generated as byproducts of the cochlear amplifier. These are measurable in the auditory canal and represent an objective method for diagnosing functional disorders of the inner ear. Conventional DPOAE and TEOAE methods permit detection of hearing impairment, but with less than desirable accuracy. OBJECTIVE: By accounting for DPOAE generation mechanisms, the aim is to improve the accuracy of inner-ear diagnosis. MATERIALS AND METHODS: DPOAEs consist of two components, which emerge at different positions along the cochlea and which may cause artifacts due to mutual interference. Here, the two components are separated in the time domain using short stimulus pulses. Optimized stimulus levels facilitate the acquisition of DPOAEs with maximum amplitudes. DPOAE and Békésy audiograms were recorded from 41 subjects in a clinically relevant frequency range of 1.5 to 6 kHz. RESULTS: The short stimulus pulses allowed artifact-free measurement of DPOAEs. Semilogarithmic input-output functions yielded estimated distortion product thresholds, which were significantly correlated with the subjectively acquired Békésy thresholds. In addition, they allowed detection of hearing impairment from 20 dB HL, with 95 % sensitivity and only a 5 % false-positive rate. This accuracy was achieved with a measurement time of about 1-2 min per frequency. CONCLUSIONS: Compared to conventional DPOAE and TEOAE methods, separation of DPOAE components using short-pulse DPOAEs in combination with optimized stimulus parameters considerably enhances the accuracy of DPOAEs for diagnosing impairment of the cochlear amplifier.

[Sound and velocity DPOAEs : Technology, methodology and perspectives]. / Schall- und Geschwindigkeits-DPOAE : Technologie, Methodik und Perspektiven.

Recent publications show that DPOAE measurements can generate a more accurate diagnosis, if (1) their fine structure is suppressed, and (2) if the calibration of the sound field is improved. Reduction of the fine structure is particularly important in the frequency range below 4 kHz in subjects with intact cochlear amplifier and can reduce the standard deviation of threshold estimations based on DPOAE-input/output functions from 11 dB to 6 dB. Improving the sound-field calibration has most impact in the frequency range above 4 kHz. Threshold estimations based on laserinterferometrically measured DPOAE input-output functions where the sound field was calibrated close to the tympanic membrane have been shown to reduce the standard deviation down to 8.6 dB in humans and 6.5 dB in guinea pigs. Compared with conventional DPOAE measures, such as amplitude or signal-to-noise ratio, threshold estimation based on DPOAE-I/O functions has the advantage that its slope provides additional information about the middle-ear; however, its specificity is limited. In the future, combined methods such as acoustic reflectance or laser vibrometry on the umbo promise a reliable assessment of the middle-ear contribution to DPOAE.

[Laser Doppler vibrometric measurements of DPOAE in humans. Eardrum vibrations reflect middle- and inner-ear characteristics]. / Laser-Doppler-vibrometrische Messungen von DPOAE an Menschen. Trommelfellschwingungen spiegeln Mittel- und Innenohrcharakteristika wider.

BACKGROUND: Up to now, laser interferometric vibration measurements of the human eardrum have not provided any information about cochlear function, because the measurement devices have not been sufficiently sensitive. METHODS: After designing a new type of laser Doppler vibrometer (LDV) that allows detection of displacement amplitudes down to about 1 pm, we used this device in 20 subjects to measure growth functions of the distortion products of otoacoustic emissions (DPOAE) as vibrations of the umbo. For comparison, DPOAE growth functions were also measured conventionally with an acoustic probe in the closed external auditory meatus. Hearing thresholds were estimated from both sets of measurements and compared with Békésy thresholds. RESULTS: The standard deviation of the threshold estimate obtained from the vibration DPOAEs was 8.6 dB, which is significantly smaller than that of the threshold estimate (16.7 dB) obtained from the acoustic DPOAEs. We attribute the smaller standard deviation for the LDV data to the fact that these measurements are made in an open sound field and are therefore less susceptible to pressure calibration errors. CONCLUSIONS: Being relatively free of sound-field measurement artefacts, the LDV method allows precise estimation of the hearing threshold. Vibration measurements of the umbo have, therefore, considerable potential for the differential diagnosis of mechanical dysfunction of the middle and inner ear.

Distortion product otoacoustic emissions measured as vibration on the eardrum of human subjects.

It has previously not been possible to measure eardrum vibration of human subjects in the region of auditory threshold. It is proposed that such measurements should provide information about the status of the mechanical amplifier in the cochlea. It is this amplifier that is responsible for our extraordinary hearing sensitivity. Here, we present results from a laser Doppler vibrometer that we designed to noninvasively probe cochlear mechanics near auditory threshold. This device enables picometer-sized vibration measurements of the human eardrum in vivo. With this sensitivity, we found the eardrum frequency response to be linear down to at least a 20-dB sound pressure level (SPL). Nonlinear cochlear amplification was evaluated with the cubic distortion product of the otoacoustic emissions (DPOAEs) in response to sound stimulation with two tones. DPOAEs originate from mechanical nonlinearity in the cochlea. For stimulus frequencies, f1 and f2, with f2/f1 = 1.2 and f2 = 4-9.5 kHz, and intensities L1 and L2, with L1 = 0.4L(2) + 39 dB and L2 = 20-65 dB SPL, the DPOAE displacement amplitudes were no more than 8 pm across subjects (n = 20), with hearing loss up to 16 dB. DPOAE vibration was nonlinearly dependent on vibration at f2. The dependence allowed the hearing threshold to be estimated objectively with high accuracy; the standard deviation of the threshold estimate was only 8.6 dB SPL. This device promises to be a powerful tool for differentially characterizing the mechanical condition of the cochlea and middle ear with high accuracy.

[Electromechanical transduction: influence of the outer hair cells on the motion of the organ of Corti]. / Elektromechanische Transduktion: Einfluss der äusseren Haarsinneszellen auf das Bewegungsmuster des Corti-Organs.

BACKGROUND: The somatic electromotility of the outer hair cells can be induced by an extracellular electrical field. This enables us to investigate the electromechanically induced motion of the organ of Corti. METHODS: The electrically induced motion of the guinea-pig organ of Corti was measured with a laser Doppler vibrometer in three cochlear turns at ten radial positions on the reticular lamina (RL) and six on each of the upper and lower surfaces of the tectorial membrane (TM). RESULTS AND CONCLUSIONS: We found a complex vibration pattern of the RL and TM, leading to a stimulus synchronous modulation of the depth of the subtectorial space in the region of the inner hair cells (IHCs). This modulation causes radial fluid motion inside the space up to at least 3 kHz. This motion is capable of deflecting the IHC stereocilia and provides an amplification mechanism additional to that associated with basilar-membrane motion.

[Laser Doppler vibrometry: a new tool for diagnosing hearing loss with an intact eardrum]. / Laser Doppler vibrometria: un nou instrument pentru diagnosticarea Hipoacuziei cu timpan închis.

The diagnosis of hearing loss with an intact eardrum frequently requires an entire battery of hearing tests, without the guarantee of an exact diagnosis. The techniques frequently provide only orientation for it, without establishing the site of the lesion and the etiology of the hearing loss. Laser Doppler vibrometry is a new technique, which has recently proved capable, of partially resolving this problem. The method is based on the study of the sound-induced vibration of the eardrum in humans in vivo, using a laser Doppler vibrometer. The method proved to be useful in the diagnosis of the pathology of the middle ear sound transmission system, avoiding the need for exploratory tympanotomy. Called "laser-audiometry", the method promises to become a new diagnostic tool for hearing impairment.

Pronounced infracuticular endocytosis in mammalian outer hair cells.

Endocytosis in cochlear hair cells was investigated by staining with the vital fluorescent dye FM 1-43, that partitions reversibly into membranes and is trapped in vesicles during endocytosis. The temporal development and spatial distribution of FM 1-43 induced fluorescence was investigated using confocal laser-scanning microscopy. FM 1-43 rapidly and intensely stained cochlear hair cells, leaving the supporting cells unstained. For short application (0.2-30 s), only the infracuticular region of outer hair cells (OHCs) was labeled, whereas for long application (30-60 s), the OHCs were also labeled in the infranuclear zone and along a central strand extending from the infracuticular zone down to the nucleus, as well as along the entire cell membrane. Except for the cell membrane, the infracuticular zone, directly below the cuticular plate, showed the most rapid and intense staining, and in most cases staining was spherically shaped with a diameter of 3-7 microm. Localization and size of this infracuticular staining coincided with Hensen's body, a specialized variant of the endoplasmic reticulum. In contrast to the OHCs, apical fluorescence of inner hair cells presented a homogeneous distribution. When OHCs were incubated in FM 1-43 for longer than 1 min, many points of contact between the central strand, the infracuticular zone and the lateral cell membrane were observed. Since Hensen's bodies are a specialty of OHCs and the fluorescent staining pattern of these cells was unique, it is proposed that Hensen's body is involved in the turnover of OHC-specific proteins, such as those involved in the molecular machinery of the motor action of the plasma membrane.

Reciprocal electromechanical properties of rat prestin: the motor molecule from rat outer hair cells.

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Distortion product otoacoustic emissions in human hypercholesterolemia.

Epidemiological and experimental studies suggest that hypercholesterolemia promotes the development of sensorineural hearing loss; however, the underlying cellular pathomechanism remains obscure. In the present study, 20 healthy subjects and 20 patients with familial hypercholesterolemia were compared with respect to their hearing function. None of the 40 persons reported any history of hearing disorder. In accordance with this subjective impression, mean hearing thresholds were within the normal, age-dependent ranges in both groups. In contrast, the single-generator distortion product otoacoustic emissions (sgDPOAE) were reduced at and above 4 kHz. Input-output functions of DPOAE could be subdivided into three groups: (i) normal, with unity slope at low intensities and slope less than unity (0.24+/-0.07 dB/dB at higher intensities; (ii) pathologic, described by a single straight line; (iii) ill-defined, with data usually indistinguishable from the background noise level. The ill-defined DPOAE behavior was only found in patients with hypercholesterolemia; namely, for 25% of patients at f(2)=1.5 kHz and for 50% at f(2)=4 kHz. Patients belonging to the pathologic and ill-defined DPOAE groups had significantly (P<0.05) higher total serum cholesterol and LDL-cholesterol levels compared with subjects from the normal DPOAE group. While hearing thresholds of patients with ill-defined growth functions were not statistically different from those of normal subjects, speech scores were significantly reduced in these cases. The data imply that nonlinear mechanical processes in the cochlea are compromised in hypercholesterolemic patients.

[Physiological effects of destruction of the tip links of cochlear hair cells. Significance for noise-induced hearing loss]. / Physiologische Auswirkungen einer Zerstörung der Tip-Links kochleärer Haarsinneszellen. Bedeutung für die lärminduzierte Schädigung de Gehörs.

Sound overexposure is known to cause damage to cochlear structures and can induce permanent or temporary hearing loss and tinnitus. Perhaps the most sensitive of these structures to sound overexposure are the tip links. In this paper the electrophysiological effects of pharmacological destruction of the tip links of outer hair cells was investigated. Outer hair cells treated with elastase (20 U/mL) or BAPTA (5 mM) no longer responded to sinusoidal stimuli. In contrast to common belief, transduction channels opened due to loss of tip links. Such opened channels can allow K+ and Ca2+ to enter the cell from the endolymphatic space and cold lead to permanent depolarization. This influx of cations caused by loss of tip links, together with the subsequent hair-cell depolarization, might be a source of sensorineural hearing loss and tinnitus associated with acoustic trauma.

Three-dimensional motion of the organ of Corti.

The vibration of the organ of Corti, a three-dimensional micromechanical structure that incorporates the sensory cells of the hearing organ, was measured in three mutually orthogonal directions. This was achieved by coupling the light of a laser Doppler vibrometer into the side arm of an epifluorescence microscope to measure velocity along the optical axis of the microscope, called the transversal direction. Displacements were measured in the plane orthogonal to the transverse direction with a differential photodiode mounted on the microscope in the focal plane. Vibration responses were measured in the fourth turn of a temporal-bone preparation of the guinea-pig cochlea. Responses were corrected for a "fast" wave component caused by the presence of the hole in the cochlear wall, made to view the structures. The frequency responses of the basilar membrane and the reticular lamina were similar, with little phase differences between the vibration components. Their motion was rectilinear and vertical to the surface of their membranes. The organ of Corti rotated about a point near the edge of the inner limbus. A second vibration mode was detected in the motion of the tectorial membrane. This vibration mode was directed parallel to the reticular lamina and became apparent for frequencies above approximately 0.5 oct below the characteristic frequency. This radial vibration mode presumably controls the shearing action of the hair bundles of the outer hair cells.

Characteristics of the travelling wave in the low-frequency region of a temporal-bone preparation of the guinea-pig cochlea.

This study provides a detailed quantitative description of the acoustically evoked vibration responses in the low-frequency region of the in vitro guinea-pig cochlea. Responses of the basilar membrane, the reticular lamina and Hensen cells were measured with a laser Doppler vibrometer, without the need for introducing artificial light reflectors. The apex of the cochlea was opened, leaving the helicotrema intact. Two response components were detected: a 'fast' component, which was probably caused by the hole in the cochlea, and a 'slow' component, which shared the features of a classical travelling wave. The velocity response of the 'slow' component exhibited a relatively flat low-frequency slope (15 dB/oct) and a much steeper high-frequency roll-off (third turn: -47 dB/oct; fourth turn: -35 dB/oct). The group delay was dependent on the characteristic frequency. In the fourth turn, the sharpness of the velocity tuning curves (Q(10 dB): 1.0) was similar to those of in vivo mechanical and neural recordings, whereas in the third turn the tuning (Q(10 dB): 1.1) was much less than for in vivo recordings. The results indicate that cochlear amplification, which is responsible for the high sensitivity and sharp tuning in the basal part of the cochlea, is much less pronounced in the apical turn of the cochlea.

Evidence for active, nonlinear, negative feedback in the vibration response of the apical region of the in-vivo guinea-pig cochlea.

The transverse vibration response of the organ of Corti near the apical end of the guinea-pig cochlea was measured in vivo. For cochleae in good physiological condition, as ascertained with threshold compound action potentials and the endocochlear potential, increasing amounts of attenuation and phase lag were found as the intensity was decreased below 80 dB SPL. These nonlinear phenomena disappeared post mortem. The data suggest that an active, nonlinear damping mechanism exists at low intensities at the apex of the cochlea. The phase nonlinearity, evident at all frequencies except at the best frequency (BF), was limited to a total phase change of 0.25 cycles, implying negative feedback of electromechanical force from the outer hair cells into a compliant organ of Corti. The amplitude nonlinearity was largest above BF, possibly due to interaction with a second vibration mode. The high-frequency flank of the amplitude response curve was shifted to lower frequencies by as much as 0.6 octave (oct) for a 50-dB reduction of sound intensity; the reduction of BF was 0.3 oct, but there was no change of relative bandwidth (Q(10 dB)). Detailed frequency responses measured at 60 dB SPL were consistent with non-dispersive, travelling-wave motion: travel time to the place of BF (400 Hz at 60 dB SPL) was 2.9 ms, Q(10 dB) was 1.0; standing-wave motion occurred above 600 Hz. Based on comparison with neural and mechanical data from the base of the cochlea, amplitudes at the apex appear to be sufficient to yield behavioural thresholds. It is concluded that active negative feedback may be a hallmark of the entire cochlea at low stimulus frequencies and that, in contrast to the base, the apex does not require active amplification.

Limiting dynamics of high-frequency electromechanical transduction of outer hair cells.

High-frequency resolution is one of the salient features of peripheral sound processing in the mammalian cochlea. The sensitivity originates in the active amplification of the travelling wave on the basilar membrane by the outer hair cells (OHCs), where electrically induced mechanical action of the OHC on a cycle-by-cycle basis is believed to be the crucial component. However, it is still unclear if this electromechanical action is sufficiently fast and can produce enough force to enhance mechanical tuning up to the highest frequencies perceived by mammals. Here we show that isolated OHCs in the microchamber configuration are able to overcome fluid forces with almost constant displacement amplitude and phase up to frequencies well above their place-frequency on the basilar membrane. The high-frequency limit of the electromotility, defined as the frequency at which the amplitude drops by 3 dB from its asymptotic low-frequency value, is inversely dependent on cell length. The frequency limit is at least 79 kHz. For frequencies up to 100 kHz, the electromotile response was specified by an overdamped (Q = 0.42) second-order resonant system. This finding suggests that the limiting factor for frequencies up to 100 kHz is not the speed of the motor but damping and inertia. The isometric force produced by the OHC was constant at least up to 50 kHz, with amplitudes as high as 53 pN/mV being observed. We conclude that the electromechanical transduction process of OHCs possesses the necessary high-frequency properties to enable amplification of the travelling wave over the entire hearing range.

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti.

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

Evidence for opening of hair-cell transducer channels after tip-link loss.

The mechanosensitive transducer channels of hair cells have long been proposed to be gated directly by tension in the tip links. These are thin, elastic extracellular elements connecting the tips of adjacent stereocilia located on the apical surface of the cell. If this hypothesis is true, the channels should close after destruction of tip links. The hypothesis was tested pharmacologically using receptor currents obtained in response to mechanical stimulation of the stereociliary bundle of outer hair cells isolated from the adult guinea pig cochlea. Application of elastase (20 U/ml) or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA; 5 mM), both of which are known to disrupt tip links in other hair-cell preparations, led to the expected irreversible loss of receptor currents. However, the cells then displayed a maintained inward current, implying that channels were left permanently open. This current was similar in magnitude to the receptor current before treatment and was reduced reversibly by known blockers of mechanosensitive channels, namely, dihydrostreptomycin (100 microM), amiloride (300 microM), and gadolinium ions (1 mM). These observations suggest that the maintained current flows through the mechanosensitive channels. Electron microscopical analysis of isolated hair cells, exposed to the same concentrations of elastase or BAPTA as in the electrophysiological experiments, demonstrated an almost total loss of tip links in hair bundles that showed no evidence of other mechanical damage. It is concluded that although the tip links are required for mechanoelectrical transduction, the channels are not gated directly by the tip links.

Development of a narrow water-immersion objective for laserinterferometric and electrophysiological applications in cell biology.

Laserinterferometric studies of the micromechanical properties of the organ of Corti using isolated temporal bone preparations are well established. However, there are relatively few measurements under in vivo conditions in the apical region of the cochlea because of its inaccessibility with commonly used techniques. Recently, optical-design programs have become affordable and powerful, so that the development of an optimized optical system is within the budget of physiologists and biophysicists. We describe here the development of a long-range water-immersion objective. To circumvent anatomical constraints, it has a narrow conical tip of taper 22 degrees and diameter 2.4 mm. It is a bright-field reflected-light illumination, achromatic objective with magnification of 25x/infinity, a working distance of 2.180 mm and a numerical aperture of 0.45. Chromatic errors are corrected at 546.1 and 632.8 nm, with emphasis on the latter wavelength which is used by the laser interferometer. The field curvature is relatively flat and a diffraction limitation (Strehl ratio better than 0.8) can be obtained in a field of 0.4 mm diameter. Using this objective, sound-induced vibrations of hair cells and Hensen cells could be recorded without placing a reflector on the target area. In addition, this objective was found to be diffraction-limited in the near infra-red (750-830 nm), with a slightly different working distance (2.186 mm), making it suitable for patch-clamp experiments using infra-red, differential interference contrast.