Two-photon excitation microscopy has the potential as an effective, non-invasive, imaging tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue absorption coefficient and reduced scattering coefficient in the infrared wavelengths as compared to ultraviolet light. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multi-photon excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 100 μm. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multi-photon excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical biopsy.
