Probing Collagen Organization: Practical Guide for Second-Harmonic Generation (SHG) Imaging

Second-harmonic generation (SHG) microscopy is a powerful microscopy technique for imaging collagen and other biological molecules using a label-free approach. SHG microscopy offers the advantages of a non-linear imaging modality together with those ones of a coherent technique. These features make SHG microscopy the ideal tool for imaging collagen at high resolution and for characterizing its organization at various hierarchical levels. Considering that collagen organization plays a crucial role in fibrosis and in its development, it would be beneficial for the researcher working in the field of fibrosis to have a manual listing crucial points to be considered when imaging collagen using SHG microscopy. In this manuscript, we try to answer this demand by providing state of the art protocols, methods and laboratory tips related to SHG microscopy, and by discussing advantages and limitations of the use of SHG for studying fibrosis.