Second harmonic generation light quantifies the ratio of type III to total (I plus III) collagen in a bundle of collagen fiber

The ratio of type III to type I collagen is important for properly maintaining functions of organs and cells. We propose a method to quantify the ratio of type III to total (type I + III) collagen (lambda(III)) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the lambda(III) of collagen gels was examined, and the slope (k(1)) and SHG light intensity at 0% type III collagen (k(2)) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k(3)) of the relationship was determined. The lambda(III) in a collagen fiber bundle was estimated from these constants (k(1-3)) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained lambda(III) = 84.7% +/- 13.8% and lambda(III) = 17.5% +/- 15.2%, respectively. These values concurred with those obtained with a typical quantification method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings demonstrated that the method proposed is useful to quantify the ratio of type III to total collagen in a collagen fiber bundle.