To observe and perform a cell count with D+:
Submerge or fill the endothelial side of cornea with either Sucrose (1.8%) or BSS to swell the borders and do count cells. Sucrose is superior to BSS (according to Amy Rigor of Australis Scientific, Syndney, Australia). Sucrose gets faster and more obvious results.
It’s very important to match as close as possible the osmolarity of media to deswelling solution
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Molecular weight of Dextran-366 g/mole
Mw of BSS- 679.3
Mw of Sucrose- 342.3
You need to capture the Enhance image of swelled border into Specular box for analysis. Since widening of the border is the transient phenomena and only last 10-15 minutes, Enhance-mode should be ready to capture prior to submerge cornea to BSS or Sucrose.
You can scan entire cornea (limbus-to-limbus) using X-Y knob on the stage. In the area devoid of cell pattern (dark area- bottom left-side) damaged cells are shown by Trypan Blue staining.
If you would like to evaluate damages (Not cell count), you can use the Enhance-mode, looking at the apical surface of the endothelium (Even cornea is as thick as 1000-1500um) during the organ culture process, if cells ate normal and not compromised, surface will be very smooth. If cells are damaged, you see 3-dimensional structures.
For this slide, the bottom right image, the surface of endo is fairly smooth indicating this cornea is normal. Diagonal multi-lines you see are the collagen fibers, while the top right images shows many 3D damaged structures. D+ also has low magnification Finder mode to allow you to see the entire cornea like a slit lamp image (top left).
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