Correlative multiphoton microscopy and infrared nanospectroscopy of label-free collagen

Collagen is the most abundant protein in mammals and represents the main component of connective tissues, such as skin, cornea, artery or tendon. The three-dimensional multiscale organization of collagen is highly specific to every tissue and directly determines its physical and mechanical properties. This project aims at developing a new analytical method for in situ mapping of the fibrillar and denatured collagen multiscale structure in label-free biological tissues. To address this issue, infra-red nanospectroscopy (AFM-IR), which enables chemical mapping at nanometer scale, is combined to multiphoton microscopy based on Second Harmonic Generation (SHG) and 2PEF (two-photon excited fluorescence) signals, which probes collagen structure at micrometer scale. Optical signatures from multiphoton microscopy show that fibrillar collagen exhibits strong SHG signals and gelatin emits fluorescence signals. AFM-IR analysis shows that IR spectra exhibit amide I band and only in the case of gelatin an absorbing band around 1730 cm^-1. Correlation of both techniques before and after denaturation on the same samples confirms this optical and chemical signatures of gelatinization process. The correlative imaging of IR nanospectroscopy and multiphoton microscopy of fibrillar collagen and gelatin structural states provide a calibration of the multiphoton signals that can be further used for the assessment of degradation of the collagen within tissues such as cornea or skin due to injuries or diseases.