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V. Bantseev, D. McCanna, J.-Y. Driot, K. W. Ward, J. G. Sivak; Distribution of Organelles Identified in Epithelium of Rat Corneas in vivo Using Confocal Laser Scanning Microscopy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4020.
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The purpose of this study was to investigate the feasibility of using confocal laser scanning microscopy together with specific fluorescent markers to assess the distribution of cellular organelles in corneal epithelium of rat in vivo.
Following anaesthesia the corneal surface of the eyes of anaesthetized rats was stained in vivo with a combination of the cell-permeable mitochondria (Rhodamine 123, 20µM final concentration) and nuclear-specific dye (Hoechst 33342, 10µg/ml). Animals (n=5) were then placed on a custom-designed heated microscope stage (to maintain body temperature) with the cornea facing glass-bottom multi-well plates (MatTek Corp., Ashland, MA). High resolution optical stacks (up to 10µm) and time series (up to 5min.) of corneal epithelium were acquired with a Zeiss 510 Meta 18 confocal laser scanning microscope (CLSM, Carl Zeiss Canada Ltd., Toronto, ON) system equipped with an inverted Axiovert 200M microscope and 40-x water-immersion C-Apochromat high numerical aperture objective (NA 1.2) and appropriate lasers and emission filters. Subsequent image analysis was carried out using commercial LSM510 VisArt/Physiology (Carl Zeiss Canada Ltd., Toronto, ON) and MatLab software packages (Mathworks Inc., Natick, MA).
Rhodamine 123 fluorescence representing metabolically-active mitochondria could be seen in the epithelium. The distribution pattern of the mitochondria of corneal epithelial cells could be identified as heterogeneous structures ranging from less then 1µm to about 5µm in length, forming a dense calyx around the nuclei. Normal nuclear morphology (labelled with Hoechst 33342) could be seen in the superficial epithelium, with some cells exhibiting irregular nuclear morphology. Moreover, the mitochondria of sensory corneal epithelial nerves could also be identified.
This study demonstrates the feasibility of using high resolution research confocal laser scanning microscope together with specific fluorescent markers to provide a new approach to study organelle distribution in rat corneal epithelium in vivo.
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