Two-photon fluorescence and confocal video microscopy of in-vivo human skin

We present a system for in-vivo skin imaging consisting of a two-photon fluorescence and a confocal reflectance video microscope. The two devices share one microscope objective, but have separate light sources, detectors, scan-units and control electronics. This makes it possible to image a region of the skin using the two different modalities. while exploiting the specific advantages of each method. In the images we clearly distinguish several skin layers, i.e., stratum corneum, viable epidermis, basal layer and upper dermis. Close to the skin surface individual keratinocytes in the stratum corneum are visible with the two-photon microscope, while in reflectance images the texture is much more uniform. Slightly deeper in the skin the smooth cellular structure of the epidermis becomes visible for both imaging modalities. Below the basal layer, which marks the boundary between epidermis and the dermis, fibrous structure appears which can be attributed to capillary vessels and dermal collagen. We use the combined imaging device for studying the effect of occlusion on human skin. From the fluorescence images we observe swelling of the stratum corneum, while the reflectance microscope shows changes in the scattering properties due to hydration We show that by combining two microscopes in one we can obtain images that contain complementary information, thereby enhancing the potential for each individual modality.