Quantitative Analysis of Collagen Produced by Rabbit Keratocytes using Second Harmonic Generation Microscopy.

Purpose: To quantitatively analyze the collagen produced by rabbit keratocytes treated with mitomycin C (MMC) and/or transforming growth factor-beta (TGF-(1)), and to investigate the collagen arrangement in situ, using second harmonic generation (SHG) microscopy.Methods: Keratocytes were divided into four groups: The keraotocytes in group rabbit keratocytes were treated with serum free medium containing 1% methylcellulose as controls. The cells in group TGF were received with TGF-(1) (at 2 ng/ml) and the same medium as the control. After exposure to MMC at 0.02 mg/ml for 5 minutes, the cells in group MMC were received with the same treatment as controls. In group TGF-MMC, the cells were exposed to MMC at 0.02 mg/ml for 5 minutes first, and then treated as group TGF. The signals of collagen were detected by SHG at 2 and 4 weeks post-treatment. The collagen content, as well as the keratocyte numbers, was calculated by MetaMorph Offline.Results: The collagen arrangement could be detected clearly by SHG. The cell number of group TGF was higher than other groups at 2 and 4 weeks post-treatment. The collagen content in group MMC was the least among all the groups at 2 weeks, while the collagen content in group TGF was higher than in group rabbit keratocytes and MMC at 4 weeks. No significant SHG side effects were found in cell proliferation and collagen deposition.Conclusions: SHG can provide a highly sensitive method to quantitatively measure collagen content, as well as to analyze its arrangement in situ.