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Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near-infrared spectral range (680-1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti-Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes-related skin disorders in the near future., (© 2015 Wiley Periodicals, Inc.)

Nonenzymatic glycation is a multistep, slow reaction between reducing sugars and free amino groups of long-lived proteins, which affects the structural and mechanical properties of collagen-rich tissues via accumulation of advanced glycation end products (AGEs). Dental collagen is exposed to glycation as part of the natural aging process. However, in case of chronically high blood glucose, the process can be accelerated, resulting in premature stiffening of dentin, leading to tooth fragility. The molecular mechanisms whereby collagen glycation evokes the loss of mechanical stability in teeth are currently unknown. In this study, we used 2-photon and atomic force microscopies to correlate structural and mechanical changes in dental collagen induced by in vitro glycation. Young tooth samples were demineralized and cut longitudinally into 30-µm sections, then artificially glycated in 0.5 M ribose solution for 10 wk. Two-photon microscopy analysis showed that both the autofluorescence and second harmonic–generated (SHG) signal intensities of glycated samples were significantly greater than those of the controls. Regarding the structural alteration of individual collagen fibers, a remarkable increase could be measured in fiber length of ribose-treated sections. Furthermore, nanoindentation of intertubular dentin regions revealed significantly higher stiffness in the ribose-treated samples, which points at a significant accumulation of AGEs. Thus, collagen glycation occurring during sustained exposure to reducing sugars leads to profound structural and mechanical changes in dentin. Besides the numerous oral complications associated with type 2 diabetes, the premature structural and mechanical deterioration of dentin may also play an important role in dental pathology. [ABSTRACT FROM AUTHOR]