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Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues

Jonathan A. Palero ^*, Henriëtte S. de Bruijn , Angélique van der Ploeg van den Heuvel , Henricus J. C. M. Sterenborg  and Hans C. Gerritsen ^*

^* Molecular Biophysics, Utrecht University, Utrecht, The Netherlands; and Center for Optical Diagnostics and Therapy, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Correspondence: Address reprint requests to Jonathan A. Palero, Molecular Biophysics, Utrecht University, Utrecht, The Netherlands. E-mail: j.palero{at}phys.uu.nl.

The deep tissue penetration and submicron spatial resolution of multiphoton microscopy and the high detection efficiency and nanometer spectral resolution of a spectrograph were utilized to record spectral images of the intrinsic emission of mouse skin tissues. Autofluorescence from both cellular and extracellular structures, second-harmonic signal from collagen, and a narrowband emission related to Raman scattering of collagen were detected. Visualization of the spectral images by wavelength-to-RGB color image conversion allowed us to identify and discriminate tissue structures such as epidermal keratinocytes, lipid-rich corneocytes, intercellular structures, hair follicles, collagen, elastin, and dermal cells. Our results also showed morphological and spectral differences between excised tissue section, thick excised tissue, and in vivo tissue samples of mouse skin. Results on collagen excitation at different wavelengths suggested that the origin of the narrowband emission was collagen Raman peaks. Moreover, the oscillating spectral dependency of the collagen second-harmonic intensity was experimentally studied. Overall, spectral imaging provided a wealth of information not easily obtainable with present conventional multiphoton imaging systems.