Direct Evidence for Spherical Multilamellar Bodies as Sources of Excess Light Scattering in Human Age-Related Nuclear Cataracts

Abstract

Purpose: : To determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 µm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming a different RI for the MLB and cytoplasm. The goal was to test this assumption by quantifying relative RI values of specific MLBs in electron micrographs. Methods: : Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with uranyl acetate and/or osmium tetroxide prior to dehydration and embedding in epoxy or acrylic resins. In 70 nm thin sections MLBs were imaged at 60 kV unstained (no grid stain) with a CCD camera on a Tecnai 12 TEM. Integrated intensity in digital electron micrographs was related directly to protein density, which is linearly related to RI. The relative RI of the MLB interior was calculated assuming a cytoplasmic RI of 1.42. Results: : Relative RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have RIs similar to the cytoplasm whereas others had significantly higher RIs. The higher RI MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. Membrane loss appeared to be part of the MLB maturation, producing large white spaces around dense MLB cores, especially in advanced cataracts from India. Predicted scattering from Mie plots was very high in both particles with thin rims and wide low-density rims. Conclusions: : A relatively simple and reliable method is described for obtaining an important parameter for light scattering calculations. The measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. For early stage nuclear cataracts having 4,000 MLBs/mm3 reported previously, the forward scattering could be more than 30% of the incident light.