The capability of the THG enhancement in vivo was evaluated by conducting studies in animal models and human. Previous reports have indicated that tattoo pigments in human skin stain the cytoplasm of fibroblasts deep in the dermis layer.38,39 Tattoo dyes thus serve as in vivo THG contrast agents of fibroblasts in mouse and human skin in our studies. For a fair comparison, the intensities of the in vivo THG images were obtained under the same PMT voltage. The experimental setup of the mouse skin in vivo and human skin in vivo can be found in our previous reports.13,21 For animal studies, the experimental protocols were approved by the National Taiwan University Institutional Animal Care and Use Committee (NTU-IACUC). We used seven-week-old female imprinting control region mice. We tattooed the undiluted black dye to the mouse skin and performed in vivo HGM on the tattooed skin after six months. Figure 3(a) and 3(b) shows examples of the horizontally sectioned SHG (green) and THG (purple) images at a depth of 30 μm below the skin surface taken from the dermal layer of the normal and tattooed mouse skin, respectively. Compared with the normal mouse skin [Fig. 3(a)], the enhanced THG signals from the tattoo pigments [Fig. 3(b) (arrows)], which provides the contrast of fibroblasts,38,39 was revealed within the collagen network. The contrast of the collagen network was provided by the SHG signals. In addition, in vivo HGM was also performed on human tattooed skin. The image acquisition process was performed under the informed consent approved by the Research Ethics Committee of the National Taiwan University Hospital. A volunteer with black tattoo was included in this study. The safety of the in vivo HGM imaging for human skin has been demonstrated previously.21,25,26 The tattooed volunteer felt no pain nor had other unpleasant feeling during and after the experiments. The observed site was also immediately examined by a physician after in vivo observation to check for clinical adverse symptoms. No skin changes can be found on the observed sites. We do not know the content of the black tattoo dye from the tattooed volunteer. However, results similar to those from the in vivo animal study were also obtained from the horizontally sectioned HGM images of the dermal layer in the normal and tattooed skin. The HGM images of the normal skin [Fig. 3(c)] were taken from the untattooed skin adjacent to the tattooed skin at a depth of 60 μm below the skin surface. The strongly enhanced THG signals were again observed in the tattooed region in the dermis layer, providing the contrast of fibroblasts,18,19 within the collagenous network as shown in Fig. 3(d) (arrows). Fibroblasts are associated with cancer cells at all stages of cancer progression,40 and their structural and functional contributions need to be studied in detail. Our reported in vivo study thus indicates the potential of the tattoo dye as an in vivo THG contrast agent to specify the fibroblast in human and animal skin for clinical and preclinical imaging.