Comparison of SEM processing methods for cultured human lens epithelial cells grown on flat and microcarrier bead substrates.
The increasing importance of in vitro models has presented new challenges in SEM processing techniques. The present study has evaluated the quality of preservation of cultured human lens epithelial cells processed by critical point, Peldri II, and tert-butyl alcohol drying. Specimens processed by critical point drying produced specimens with severe cracking of cell processes and microcracks across cell membrane surfaces. Peldri II and tert-butyl alcohol drying eliminated breakage of the filopodia and lamellipodia as well as eliminating the microcracks across the apical membrane surface. The morphology of lens epithelial cells grown on Cytodex 3 beads appeared rounded with convoluted membrane surfaces. These morphological features were present for cells processed by all three methods. Cytodex 3 beads were subsequently shown to shrink 52% in diameter during dehydration, which results in an 89% reduction in volume for the bead. Cells grown on Biosilon beads, which do not shrink, had a morphology similar to the cells grown on a flat substrate. These results indicate that Peldri II and tert-butyl alcohol drying offer an attractive alternative to critical point drying when preparing cultured cells for SEM. Interpretation of cultured cell morphology must consider shrinkage of the substrate material as a possible contributor to artifact.
Cantu-Crouch
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Alcon Laboratories, Inc., Fort Worth, Texas 76134, USA.
Howe
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McCartney
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MD
D000328:Adult; D000440:Butanols; D002478:Cells, Cultured; D003890:Desiccation / Q000379:methods; D004848:Epithelium / Q000648:ultrastructure; D005466:Fluorocarbons; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008863:Microspheres; D020002:tert-Butyl Alcohol
D000440:Butanols; D005466:Fluorocarbons; C062793:Peldri II; D020002:tert-Butyl Alcohol