Optical Metabolic Imaging of Human Skin Using Multiphoton FLIM Tomography

Optical metabolic imaging (OMI) of human skin is based on the cellular autofluorescence, mainly due to two photon excitation of fluorescent coenzymes such as NAD(P)H (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), with a near infrared femtosecond laser at high MHz repetition rate. Intratissue mitochondria can be imaged based on coenzyme fluorescence with a lateral resolution of less than 500 nanometers. Interestingly, a specific single cell deep in the skin can be traced and imaged for hours without motion artifacts using a magnetic interface. Studies with a tunable Ti:sapphire laser for NAD(P)H as well as flavins excitation have been conducted on patients suffering from dermatitis, psoriasis, chronic wounds, and cancer. OMI has also been performed on volunteers during oxygen inhalation as well as on astronauts prior and after long-term space flights. “Energizing” cosmetics with oxygen scavengers was found to change NAD(P)H levels in the skin volunteers. Of particular interest of OMI of human skin is the change of the ratio of free NAD(P)H to bound NAD(P)H as an indicator of altered cellular metabolism that can be determined by autofluorescence lifetime imaging and by using a single excitation wavelength such as 780 nanometers. The fluorescence lifetime of the protein-bound nicotinamide coenzyme is nearly one order larger than the free, non-bound form. Using a biexponential approach, the mean autofluorescence lifetime and the metabolic ratio “short fluorescent component to long fluorescent component” as OMI parameters can be depicted as false colored stacks of optical sections through the epidermis. In conclusion, two-photon autofluorescence lifetime imaging (“metabolic FLIM”) provides a powerful functional imaging modality for dermatology, pharmacy, and cosmetic research to realize high-resolution optical metabolic imaging of human skin.