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Giuseppe Lombardo, David Merino, Pablo Loza-Alvarez, Marco Lombardo, ; Corneal stroma collagen order determined by polarization sensitive second harmonic microscopy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4858.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate stromal collagen order distribution across depth in donor human corneas.
Eight human donor corneas were imaged via polarization sensitive second harmonic generation microscopy (P-SHG). Each corneal tissue was cut in two lenticules using an eye-bank Moria microkeratome with 200 µm head. A linear polarizer, followed by a zero order λ/2 wave plate placed on a motorized rotational stage, was used to control on the fundamental input polarization at the sample plane. Forward SHG images were collected from anterior and posterior lenticules and used for subsequent analysis of stromal collagen structural organization. Under assumption of cylindrical symmetry of collagen fibers and using mathematical tensorial framework, a biophysical model was developed to quantify the mean average fiber orientation and the degree of fiber order orientation (order parameter, S) across corneal depth. The S parameter indicates how clustered the collagen fibers are, and whether they are clustered along the main average fiber direction (S≥0) or orthogonal to it (S<0). For each pSHG image obtained across the stroma, the percentage of pixels with a negative order parameter value was recorded (inhomogeneity ratio, N). Logistic regression analysis was performed to classify stromal collagen order of the anterior and posterior corneal lenticules based on S and N variables. Receiver operating characteristic (ROC) curves were plotted to obtain critical values that allow classification with maximum accuracy.
The mean S values of the anterior and posterior stromal lenticules were 0.49 ± 0.03 and 0.57 ± 0.04 respectively. The mean N values of the anterior and posterior stromal lenticules were 21.0 ± 0.8% and 16 ± 1.10% respectively. Regression logistic analysis reached an area under the ROC curve of 1.00, showing a sensitivity and a specificity of 100% to discriminate between anterior and posterior stromal collagen structures.
P-SHG was shown to be an accurate method to ascertain structural order of stromal collagen across corneal depth. Further studies are needed to understand the reliability of the technique to screen corneal disorders, such as keratoconus, and the efficacy of treatment aiming to manipulate collagen structural organization.
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