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Chiroptical Effects in the Second Harmonic Generation from Collagens I and IV: Applications in Nonlinear Microscopy
A.-M. Pena, T. Boulesteix, T. Dartigalongue, M. Strupler, E. Beaurepaire and M.-C. Schanne-Klein

An emerging application of multiphoton microscopy is the observation of unstained intact biological tissues based on intrinsic sources of nonlinear signals. However, reliable biomedical imaging requires a thorough analysis of these endogenous signals. Looking at skin biopsies, we focused on 2-Photon Excited Fluorescence (2PEF) arising from cytokeratins in the epidermis, and second harmonic generation (SHG) from collagen fibers in the dermis. We determined the 2PEF excitation spectrum and action cross section of purified keratins from human epidermis, and obtained a good agreement with in situ measurements. In order to analyze the role of chirality and collagen type in SHG signal, we performed polarization-resolved surface SHG experiments on thin films of collagens I and IV molecules. We observed that collagen I and IV molecules exhibit comparable SHG signals, except for the chiral contributions which are absent for collagen IV and represent typically 50% of the signal for collagen I. We concluded that the large collagen I SHG efficiency is dominated by coherent effects due to the high density and quasi-crystalline order in collagen fibrils, whereas the lack of any alignment within the collagen IV molecules explains the absence of signal from this collagen type in SHG microscopy.

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