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M.J. Costello, K.O. Gilliland, K.L. Leonard, W.C. Fowler, S. Johnsen; Confocal and Multiphoton Imaging of Human Nuclear Membranes and Multilamellar Bodies in Transparent Lenses and Age–Related Nuclear Cataracts . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1975.
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© ARVO (1962-2015); The Authors (2016-present)
Previous morphological studies have documented the presence of rare, potential scattering multilamellar bodies (MLBs) in human lens nuclei using 1 µm thick histological sections and 70 nm thin electron microscopy sections (EER 79, 563–76, 2004). The MLBs were more numerous in age–related nuclear cataracts than in transparent donor lens nuclei and their distribution appeared to be random. The MLB sizes were corrected for the probability of a section passing through the particle diameter and the particle volume density was estimated from planar section data using a stereology formula. To improve the direct determination of quantitative data, methods have been developed to preserve and label the membranes in nuclei of aged human lenses for fluorescence scanning confocal and multiphoton imaging.
Whole donor lenses and lens nuclei after extracapsular extraction were immersion fixed in 10% formalin for 24 hrs and 4% paraformaldehyde for 48 hrs prior to Vibratome sectioning. The 160 µm thick sections were immersion stained for 24 hrs in lipid dyes, DiI or DiO, for confocal or multiphoton imaging, respectively, on a Zeiss LSM 510. Washed sections were stabilized in Permount under #1 cover slips. Inner nuclear regions were examined using a plan–apochromat 63x oil (na = 1.4) lens.
Complex membrane interfaces between nuclear fiber cells and the lipid–rich coating of MLBs were readily visualized in the confocal and multiphoton images. High quality images were taken to depths > 50 µm in confocal z–stacks that allowed reconstruction to visualize single spherical MLBs and the unusual shapes of MLBs with several components as described previously. Multiphoton imaging produced high quality images to depths > 100 µm into the Vibratome sections, thus giving access to nearly the entire volume of the lens nuclei. MLB diameters ranged from 1–4 µm and were easily distinguished from the numerous interdigitations of adjacent cells.
Initial immersion fixation stabilized membrane structures sufficiently for lipid dyes to penetrate uniformly throughout thick Vibratome sections of human lens nuclei. Fluorescent confocal and multiphoton imaging permitted clear visualization of membrane processes and MLBs. The size, shape and distribution of MLBs observed in fluorescent images were consistent with previous data from sections and the reconstruction of MLB shape from optical sections reinforced their distinctive features.
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