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A. Peral, L. Rivas, J. Pintor; Characterization of Ocular Mucins in Aniridia and Sjögren Patients by Laser Scanning Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5673.
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The visualization of human mucins in aniridia and Sjögren patients combining impression cytology samples and laser scanning microspcopy
Healthy individuals, aniridia and Sjögren patients were chosen for the study. The impression cytology samples were visualized by means a Zeiss LSM Pascal microscope. Magnifications of 20X, 40X (oil) and a scan zoom of 2 over the 40x image (≈80) were used for the visualization. LSM was performed by exciting the samples with the 546 nm wavelength laser to make PAS-hematoxilyn produce red fluorescence. Stacking images were taken to generate 3-D images suitable to observations, measurements and quantifications. The impression cytology was carried out after administration of a topical anaesthesia. Strips of cellulose acetate filter paper (Millipore HAWP304) of 5x5mm were applied on the conjunctiva for 3 seconds. The filter material was fixed in 96% ethanol, stained with PAS-hematoxylin, dehydrated in ascending grades of ethanol, then xylene and permanently mounted.
Light microscopy of impression cytology samples permits to observe epithelial (pink) and goblet cells (reddish purple). The excitation of pigment with the laser permitted us to identify epithelial, goblet cells and mucins. Performing a stack followed by a 3-D reconstruction, made possible to see the mucins surrounding goblet cells forming a cloud above them. This reconstruction allowed the measurement of the mucin cloud height -MCH- over the cell layer for the control (n=25, MCH=6.81±1.80 µm) and the aniridia samples (n=20, MCH=2.55±1.00 µm), P-value <0.0001. Although no goblet cells were found in the Sjögren samples (n=10), it was observed a big amount of mucin over the non-secretory epithelial cells. All the analized samples were from the interpalpelbral region .
Laser scanning microscopy technique to visualize mucins may help to estimate other characteristics and to better understand ocular surface pathologies where the amount or the quality of these proteins could be altered.
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