Imaging the organ of Corti in vitro and in vivo

Abstract: Recent developments in imaging techniques, namely confocal laser scanning microscopy, offer new tools in exploration of the structure and function of the inner ear. The optical sectioning property of the confocal microscope enables investigation of living cells deeper within a labeled intact tissue. This key feature has been embraced in all three our projects, using both in-vitro and in-vivo approaches. In the first study, we have developed an in-vivo model for confocal imaging of the cochlear structure in living guinea pigs. Temporal bones have been used previously for visualization in the unfixed cochlea. There is however an inevitable time limitation in all in-vitro preparations, caused by interruption of blood and nerve supply and gradual disappearance of endocochlear potential, which occurs just few hours after temporal bone dissection. Thus in-vitro models may not faithfully reflect the whole complexity of the inquired system. In-vivo preparation is deemed to be superior to in-vitro one, due to its closeness to physiological conditions. Obviously, this approach is much more demanding, especially when depicting a delicate, bone encapsulated and fluid filled structure of the cochlea in sub-optimal imaging conditions. Apart from morphology we have also investigated pathological changes induced by acoustic overstimulation. Shortening and swelling of the hair cells which was observed, coincides with results of similar experiments performed on isolated temporal bones. For the first time, we have presented detailed confocal images acquired from the living inner ear with its blood and nerve supply still preserved. Confocal laser scanning microscopy has one substantial limitation in a relatively long acquisition time. Due to the inherent point-to-point scanning mode of the microscope, it takes up to 1 second to acquire a single standard size confocal image. This is too slow to capture a swift, oscillatory motion of the organ of Corti during sound stimulation. However, a pixel dwell time of the microscope is much shorter - in the range of microseconds. This fact was utilized in the second project, with the aim to develop a confocal imaging method to visualize and measure the rapid motion patterns of stereocilia during the sound stimulation. Using a temporal bone preparation of the guinea pig cochlea, two dimensional, high-resolution images were acquired from structures vibrating at frequencies up to several hundred Hertz. Results here show that under passive conditions, the outer hair cell stereocilia deflection is larger then in the inner hair cells. Phase relations were consistent with an idea that stimulation of inner hair cells occurs through the surrounding fluid drag. The small deflection amplitudes that we measured presume active amplification present not only in the outer hair cells but also in the inner ones. Our third project stemmed from the previously described technique, which was further improved by computing the Fourier series coefficients for each acquired pixel along the time dimension. Technical improvements resulted in faster acquisition times while using larger image format (512x512pixels) and increased sound stimulation frequency interval up to 500Hz. Data measured at different sound stimulation levels from both outer and inner hair cells at the identical regions of cochlea show, that the reticular lamina does not vibrate as a stiff structure, but is subject to deformation. This is in contrary to a notion, that the organ of Corti vibrates as one rigid structure. The most profound deformation appears to occur around outer hair cells, leading to length changes of the reticular lamina. This study provides an important insight into the passive mechanics of the organ of Corti, which seems to be optimized for stimulating inner hair cells as the primary auditory receptors.