Purchase this article with an account.
K.K. H. Svoboda, J.V. Jester, W.M. Petroll; Second Harmonic Signal Analysis Demonstrates That Avian (E17) Corneal Collagen Fibrils and Keratocytes Rotate 200 Degrees in the Anterior Stroma . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3561.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We used two–photon excitation of collagen to generate a second harmonic signal (SH) that provides spatial information concerning collagen organization in 3–dimensions. In this study we used SH to investigate collagen organization in the developing chick cornea. Methods: Collagen fiber organization in 3–dimensions was evaluated in whole chicken corneas at embryonic days 8, 14, 17, and 18 using two–photon excitation to generate SH. Corneas were dissected free of other eye structures and fixed in 4% paraformaldehyde, then rinsed and analyzed with a Leica SP2 multiphoton microscope equipped with a Ti:Sapphire laser. Using an excitation frequency of 860 nm, forward scattered SH signals were detected using a transmitted light detector equipped with infrared filters. Two dimensional images (256 x 256 pixels) were obtained from the epithelium to the endothelium at 1 or 2 micron steps for analysis using Metamorph Software. Rotational angles were calculated through the thickness of the corneas. Additional corneas were stained with phalloidin and/or propidium iodide to locate other cellular structures with two–photon excited fluorescence confocal microscopy. Results: SH signals from unstained corneal tissues showed a cross hatched pattern of orthogonally arranged collagen bundles that were consistently 90 degrees apart. The 8 day corneas did not have strong SH signals and the fibrils did not appear to rotate. The 14 day corneas had more structure and the fibrils rotated 90 degrees. A strong signal was obtained from 17 and 18 day corneas that had a 200 degree clockwise rotation of collagen fibrils and cells through the anterior one–third to one–half. Very little angular rotation was detected in the posterior cornea. Phalloidin stained cells had a similar rotational pattern through the anterior cornea. Conclusions: The data suggests that stromal development in the chick involves a highly ordered orthogonal deposition of collagen fibrils that has a rotational shift in the layering of successive corneal lamellae. Further study of how this structural organization is controlled will be important to controlling matrix deposition in repair and tissue engineering.
This PDF is available to Subscribers Only