Collagen crosslink status analysed in vitro using second-harmonic generation (SHG) and fluorescence lifetime imaging (FLIM)

Vivien Lutz; Stefan Puschmann; Stefan Gallinat; Horst Wenck; Klaus-Peter Wittern; Frank Fischer

doi:10.1117/12.906455

9 February 2012 Collagen crosslink status analysed in vitro using second-harmonic generation (SHG) and fluorescence lifetime imaging (FLIM)

Vivien Lutz, Stefan Puschmann, Stefan Gallinat, Horst Wenck, Klaus-Peter Wittern, Frank Fischer

Author Affiliations +

Proceedings Volume 8207, Photonic Therapeutics and Diagnostics VIII; 820703 (2012) https://doi.org/10.1117/12.906455
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

One of the major structural proteins in human skin is collagen. Collagen and its crosslinks are essential for the mechanical stability of the skin. Looking at extrinsically aged human skin (photo damaged skin) we find a decrease of mature collagen crosslinks. Immature crosslinks an indicator of the collagen turnover are decreasing as well in extrinsically aged skin. Hence, we assume that a certain range of mature and immature crosslinks reflect a 'good quality' of collagen in terms of photoaging. In this study we established in vitro models of reduced crosslinking. We found that reduced collagen crosslinking resulted in a higher Second Harmonic Generation (SHG) intensity. Furthermore, we found a higher fibril diameter after crosslink reduction without an increase in collagen concentration. SHG is generated by a non-linear effect of femtosecond laser irradiation on collagen molecules. This effect might be influenced by the interspaces of the collagen molecules within the collagen fibril. From these findings the following hypothesis was introduced: reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril resulting in an enhanced SHG signal. Furthermore, in this study the fluorescence lifetime (FLIM) of collagen fluorescence was found to decrease in the in vitro models of reduced crosslinking. We speculate on possible mechanisms being responsible for the decrease in lifetime. Future in vivo measurements of the two parameters (SHG and FLIM) could lead to information about the collagen crosslink status, and therefore the status of photoaging of the skin.

Citation Download Citation

Vivien Lutz, Stefan Puschmann, Stefan Gallinat, Horst Wenck, Klaus-Peter Wittern, and Frank Fischer "Collagen crosslink status analysed in vitro using second-harmonic generation (SHG) and fluorescence lifetime imaging (FLIM)", Proc. SPIE 8207, Photonic Therapeutics and Diagnostics VIII, 820703 (9 February 2012); https://doi.org/10.1117/12.906455

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