Determination of collagen fibril size via absolute measurements of second-harmonic generation signals

The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea.