Thickness distribution of fresh and preserved human eardrums measured with confocal microscopy.

HYPOTHESIS: In this study, the thickness distribution of the fresh human eardrum was measured and possible thickness changes in successive stages of preservation and preparation were studied. METHODS: The thickness measurement was performed on axial fluorescence images taken perpendicularly through the membrane with a confocal microscope. The influence of fixation and preservation (in Cialit solution) on the thickness was also investigated. The same eardrum was prepared (decalcified, dehydrated, and stained) for histologic sectioning and the thickness was measured on the sections using conventional light microscopy. RESULTS: Similar thickness distributions in the measured samples (n = 3) were observed. The pars tensa has a rather constant thickness in a central region curving as a horseshoe upward around the manubrium. The membrane thickens slightly from the inferior to the superior side. The anterior region is thicker than the posterior region. In narrow bands along the manubrium, peripheral rim, and in the region inferior to the umbo, a much larger thickness in comparison with that in the central region was found. Mean thicknesses of approximately 40, 50, and 120 microm were observed in the central region of the studied eardrums, respectively. CONCLUSION: Whereas the thickness distribution of the human eardrums shows similar features, the absolute thickness seems to vary a lot from one specimen to another. There is no significant difference in thickness of the same membrane in fresh, fixed, or preserved condition. Thus, human eardrums may be safely preserved in fixative for later thickness measurements. The histologic preparation process, however, causes a significant location-dependent shrinkage.

Refractive index of tissue measured with confocal microscopy.

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382+/-0.004, at 592 nm.

Thickness of the gerbil tympanic membrane measured with confocal microscopy.

Thickness data for the gerbil tympanic membrane, an extremely thin biological membrane, are presented. Thickness measurements were performed on fresh material using fluorescence images taken perpendicular through the membrane with a commercial confocal microscope. Thickness varies strongly across the membrane. Similar thickness distributions in all samples (pars tensa n = 11; pars flaccida n = 3) were observed. The pars tensa has a rather constant thickness of about 7 microm in the central region curving as a horse shoe upwards around the manubrium. In the most superior parts of the pars tensa thickness becomes gradually twice as large. Thickness increases also steeply from the central region towards the edges (about 35 microm near the annulus and 20 microm near the manubrium). A pronounced, local thickening of about 30 microm is present close to the edge and extends as a ring along the entire annular periphery of the pars tensa. Overall, the pars flaccida is thicker than the pars tensa and has a rugged surface. Its central region has a mean thickness of about 24 microm with a mean variation of about 4 microm. The average thickness in the inferior region is slightly larger than in the superior region. The pars flaccida thickens steeply, up to about 80 microm, near the edges.

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.