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A. L. Jamil, E. Martin, K. Curtiss, T. Samuelson, P. P. Chen, M. Johnstone; Transparent Cylindrical Structures Spanning Schlemm's Canal (SC): Examination by Oblique Light, Phase Contrast, Differential Interference Contrast (Nomarski) and Fluorescence Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3214.
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© ARVO (1962-2015); The Authors (2016-present)
To examine transparent cylindrical structures spanning SC by oblique light, phase contrast, Nomarski and fluorescence microscopy.
M. Nemestrina eyes (4), 80-power dissecting microscope, HealonTM, Karnovsky’s fixative, 150 u viscoelastic cannula, fiber optic light source, phase contrast, Nomarski and fluorescence microscopy. In 4 eyes each divided into 4 quadrants introduction of viscoelastic into SC at each end of each limbal quadrant was followed by fixation. Ciliary body and iris were removed. Radial sequential 500 micron sections were cut around the entire SC circumference. Oblique illumination, phase contrast, Nomarsky and fluorescence techniques were used to image structures in SC.
Cylindrical structures spanning SC were transparent preventing identification with coaxial light at the dissecting microscope. Oblique light caused light scattering within SC structures. SC structures thus illuminated became visible when viewed against a black background placed under SC. In two M. Nemestrina eyes, 77 segments were examined and 91 cylindrical structures spanning SC photographed. Average structures/segment OD 1.33, OS 1.07. Structure frequency was 0 (15 segments) , 1 (40 segments), 2 (16 segments), 3 (5 segments) and 4 (1 segment). In Eye #8L, structures per quadrant: SN (8), IN (13), IT (14), ST (12). The short SC segments and SC dilation provided a light path to traverse the canal thus permitting phase contrast, Nomarski and fluorescence microscopy to image the transparent SC structures. The Nomarski technique with 4 z-series stacked sections (2 u /section, 690 total) demonstrated that transparent cylindrical structures spanning SC had a wall composed of endothelium enclosing a lumen connected to an open endothelial cell-lined funnel arising from SC inner wall endothelium.
In monkey eyes, transparent cylindrical structures spanning SC were not visible using standard dissecting techniques. SC dilation with viscoelastics and appropriate lighting conditions permitted SC structure identification, photographic documentation and study by phase contrast, Nomarski and fluorescence microscopy.
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