Abstract
Purpose: :
Clinical confocal scanning laser microscopes are used in vivo to examine the cornea. However, such instruments are unable to yield the precision and resolution provided by research level in vitro instruments. This study was carried out to evaluate the feasibility of using confocal laser scanning microscopy (CLSM) together with fluorescent dyes approach to provide quantitative and qualitative benchmarks of the normal distribution of cellular organelles and morphology of bovine corneal epithelium.
Methods: :
Fresh bovine eyes were obtained from a local abattoir and, in sterile conditions, 9.5mm central corneal buttons were cut using trephine blade. The corneal buttons were then placed in a glass vials containing Rhodamine 123/Hoechst 33342 (n=7), Acridine Orange (n=8) or APO#4 kit (n=6, a combination of YO–Pro–1 and propidium iodide) fluorescent dyes (Invitrogen Inc., Burlington, ON) dissolved in 10mm HBSS and incubated for 20 minutes at 370C. Following the labeling with fluorescent dyes, the corneal buttons were rinsed twice in HBSS and mounted in 1% agarose, previously dissolved in HBSS, on glass–bottom plates (MatTek Corp., Ashland, MA). High resolution X,Y (0.22µmx0.22µm) and Z–series (0.45µm) stacks of epithelial layers were acquired with a Zeiss 510 Meta 18 confocal laser scanning microscope system equipped with an inverted Axiovert 200M microscope and 40–x water–immersion C–Apochromat objective (NA 1.2). The combination of appropriate lasers and emission filters were used to visualise fluorescence. Subsequent image analysis and 3–D reconstruction was achieved using commercial LSM510 VisArt/Physiology (Carl Zeiss Ltd., Toronto ON) and MatLab software packages (Mathworks Inc., Natick, MA).
Results: :
The CLSM system allows high resolution imaging of the organelle distribution and morphology of the corneal epithelium. By using specific fluorescent markers the imaging of nuclei is possible in the superficial cells. Moreover live mitochondria can be imaged in all layers of epithelium from the superficial cells to basal cells.
Conclusions: :
Imaging of fresh bovine corneas with CLSM together with fluorescent dyes generates high resolution images. Due to the high Z resolution of less than 0.5µm, this CLSM approach enables to provide reproducible quantitative and qualitative descriptions of the normal distribution of cellular organelles and morphology of bovine corneal epithelium. Moreover, the thickness of all layers of the cornea can be precisely measured.
Keywords: cornea: basic science • cornea: epithelium • mitochondria