| This work shows that confocal microscopy allows a quantitative study of delicate 3D-biotissue in fresh condition, thus avoiding histological preparation processes. The developed procedure results in exact and accurate thickness data for mum-sized objects with a measuring error of less than 1mum. It is, however, necessary to take into account the effect of focal shift in the case of refractive index mismatch to obtain such precise data. The use of the proposed method is advised instead of the use of a paraxial approximation for the axial scale correction because the method improves measurement precision by a factor of four. The axial scaling correction factors obtained in this work show that for most practical situations the correction cannot be ignored when one wants to obtain precise quantitative data. The thickness correction method can also be used to determine with high accuracy the index of refraction of biological tissue.; The thickness measurement method was applied to fresh, untreated tympanic membranes of the gerbil, the cat and the human. Thickness had to be measured at many points as it differs strongly across the membrane. Similar thickness distributions were found in all pars tensas measured even across the species studied: (1) a very thin, central region with a rather constant thickness, curving as a horse shoe upwards around the manubrium (thickness: gerbil: about 7mum, cat: about 10mum, human: large inter-specimen variation: 40mum-120mum), (2) a thinnest zone at the inferior side, (3) a thicker zone at the supero-anterior side, (4) superior to the umbo, an anterior region thicker than the posterior region, (5) maximal thicknesses in a very small region near the entire manubrium and the entire annular periphery. The pars flaccida is found to be thicker than the pars tensa. It shows no central homogeneous zone: the thickness varies irregularly and very rapidly over short distances. Arbitrarily spaced bumps and notches are present over the entire pars flaccida surface. The thickness results advise against the use of single thickness values in mathematical models. The presented thickness and its spatial distribution can be introduced into middle ear computer models to further improve model realism. |
