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K.O. Gilliland, L. Frame, S. Johnsen, M.J. Costello; Estimates of Local Refractive Index Variations Across Micrometer Sized Particles in Human Age–Related Nuclear Cataracts . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2908.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To employ quantitative electron microscopy to determine the refractive index, which is related to protein concentration, in and around multilamellar bodies thought to be potential sources of excess forward scattering in human age–related nuclear cataracts. Methods:Based on the procedures of Lamvik and Davilla (J.Elec.Micr.Tech. 11, 97–101, 1989), who used polystyrene spheres as electron optical wedges of known mass density, we have examined evaporated carbon films of varying thickness to derive a scale of transmittance (integrated intensity) as a function of electron path length. The scale relates optical density of transmission electron micrographs to carbon concentration using the measured film thickness and the known mass density of amorphous carbon (1.9–2.0 g/cc). The optical density of micrographs of well–preserved, unstained fiber cell cytoplasm from normal and cataractous human lens nuclei were compared to the derived scale to estimate protein concentration and local refractive index. Low dose techniques were used to minimize beam damage. Results:The measurement of mass in the transmission electron microscope depends on the accurate determination of the decrease of electron flux through homogenous protein gels. Digital bright field images were used with integration of the intensity to give a reproducible measure of the electrons lost and the molecular concentration. A difficulty with thin sections is the correction for the embedding medium, such as epoxy resin, which has taken the place of water in the fixed tissue. Therefore only approximations to native refractive index are possible and the best comparisons are made within the same thin sections in adjacent regions. Cytoplasm from nuclei of human normal and cataractous lenses give similar values and are consistent with refractive index measurements of 1.40 Â± 0.01. Refractive index measurements of cytoplasm within 1–4 Âµm diameter multilamellar bodies have been found to be similar to the surrounding cytoplasm for some particles and different by 2–5% for others. Conclusions:Measurements of mass and estimated refractive index in fixed, embedded, unstained thin sections of human nuclear fiber cells suggest that the refractive index is constant among fiber cells in each developmental region. Some multilamellar bodies have been observed to have different interior refractive index, supporting the suggestion that they should be considered as potential sources of excess nuclear light scattering in cataracts.
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