Nonlinear microscopy techniques, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), are attractive solutions to this problem as they are able to probe the collagen and elastin, respectively, of the matrix in a label-free manner. Here, we posit that their combined use may contribute to enhanced diagnosis/prognosis of IPF and also further the understanding of the disease etiology and progression. SHG directly probes the structure of collagen and has been used to describe ECM alterations in several diseases, such as cancers, fibroses, and connective tissue disorders.14–27 Multiphoton microscopy of elastin has also been used for several applications, including imaging skin and cardiovascular tissues, often in conjunction with SHG and coherent anti-Stokes Raman scattering.28–30 SHG and TPEF microscopy has not yet been used extensively for lung tissues and has been limited to mouse models. For example, Abraham and Hogg31 and Pena et al.32 both have used SHG and TPEF to study the remodeling in the lung matrix in chronic obstructive pulmonary disease (COPD) and a bleomycin-mouse model of IPF, respectively. Both these studies were successful in differentiating diseased-remodeled lungs from normal lungs using a pixel-based measure of collagen coverage and a voxel ratio of the collagen/elastin balance. However, additional structural information is encoded within the collagen SHG signal that was not utilized. For example, the fiber pattern observed in the SHG images in normal and IPF tissues can be used as a machine learning classification system, enabling the collagen fibrillar pattern to be used as a label-free biomarker for IPF. This is important as, surprisingly, the fibrotic changes in the IPF matrix are considerably less characterized than the cellular aspects.