Using multiphoton microscopy (MPM), researchers at Kyoto University showed that the collagen fibers in the skin are arranged in a mesh-like lattice and not in a clear geometric orientation as previously thought. They further found that the connective tissue in the skin, known as elastic fibers, is arranged in the same way as the collagen. The study by the Kyoto team aimed to characterize the geometric organization of collagen fibers in the entire dermis layer ex vivo by using a new microscopic technique that combines MPM (the tissue clearing method) and biaxial extension. The researchers hypothesized that tissue clearing would allow observation of deep dermal layers, and biaxial extension would facilitate identification of individual collagen fibers — so the combination of these techniques would allow for quantitative analysis of fiber orientation. In previous studies, the tight packing and complex intertwining of collagen fibers has hindered accurate analysis of fiber orientation. MPM is an emerging imaging method based on nonlinear optical effects produced by near-infrared (NIR) femtosecond lasers. Second harmonic generation, a MPM imaging mode, was used to help visualize intra-organ collagen without labeling. Two-photon excited autofluorescence, another MPM imaging mode that allows for imaging without labeling, was used as well. Dynamic multiphoton microscopy images of dermal fiber architecture under biaxial tissue extension. Second harmonic generation (SHG) images show architectural changes in collagen fibers (a–c) whereas two-photon autofluorescence (TPAF) images show architectural changes in elastic fibers (d–f). λ represents the stretch ratio. Arrows of the same shape indicate the same elastic fiber. Courtesy of Kyoto University/Scientific Reports. Since labeling was unnecessary, architectural changes in fibrous networks during tissue processing could be avoided. Second harmonic generation images showed architectural changes in collagen fibers, and two-photon autofluorescence images showed architectural changes in elastic fibers. Understanding the geometric organization of fibers in the skin could lead to better insight into the mechanisms underlying human skin pliability, improving the outcome of skin grafts and transplantation. The team intends to further its research by uncovering the relationship between the collagen network and elastic tissues, and hopes that its new technique will be used to study the fibrous microarchitecture in other connective tissue networks. The research was published in Scientific Reports (https://doi.org/10.1038/s41598-019-47213-5). Collagen fibers (turquoise) are arranged in a mesh-like structure. The elastic fibers (red) also share the same architecture. Courtesy of Kyoto University/Susumu Saito.