In Vivo, Label-Free Multiphoton Microscopy Is Sensitive to Altered Metabolism and Structure in Aged Skin.

OBJECTIVE The expected increase in age-related health care costs over the coming decades underscores the need to characterize the effects of biological aging in our cells and tissues. Our objective is to use in vivo multiphoton microscopy (MPM) to find endogenous biomarkers of skin aging.

APPROACH MPM was performed on the skin of 2- and 21-month-old mice to measure the autofluorescence of metabolic cofactors, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and second-harmonic generation signal from collagen. A suite of quantitative biomarkers with sensitivity to age-related changes in bioenergetic demands, mitochondrial organization, and collagen composition and organization was evaluated using these optical sources of contrast.

RESULTS An optical redox ratio of FAD/(NADH + FAD) autofluorescence, NADH fluorescence lifetime imaging, and mitochondrial fractal dimension measurements indicated that aged keratinocytes have more fragmented mitochondria that undergo less catabolism of carbon substrates. Analysis of the dermal regions indicated that aged collagen has more nonenzymatic cross-links and has a more fragmented organization.

INNOVATION This study identifies a suite of biomarkers from label-free MPM sensitive to intrinsic aging and provides the first in vivo demonstration of sensitivity to age-dependent metabolic changes in skin through multiple independent optical parameters.

CONCLUSION Our results demonstrate that surrogate markers of metabolic function, mitochondrial organization, collagen cross-linking, and fiber organization are sensitive to a loss of homeostasis with advanced age and may be used in future studies to longitudinally assess the progression of aging.