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

Abstract

Abstract 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 (λ III ) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λ 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 λ 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 λ III = 84.7% ± 13.8% and λ 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.