Optical biopsy of in vivo human skin: Multi-photon excitation microscopy

Two-photon excitation microscopy has the potential as an effective, non-inv asive, imaging tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation sourc e 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 poin t and provides the physical basis for optical sectioning. Multi-photon exci tation microscopy at 730 nm was used to image in vivo human skin autofluore scence from the surface to a depth of about 100 mu m. The spectroscopic dat a suggest that reduced pyridine nucleotides, NAD(P)H, are the primary sourc e of the skin autofluorescence using 730 nm excitation. This study demonstr ates the use of multi-photon excitation microscopy for functional imaging o f the metabolic states of in vivo human skin cells and provides a functiona l and morphological optical biopsy.