Multiphoton Optical Image Guided Spectroscopy Method for Characterization of Collagen-Based Materials Modified by Glycation

摘要

The cross-linking with reducing sugars, known as glyca-ntion, is used to increase stiffness and strength of tissuesnand artificial collagen-based scaffolds. Nondestructivencharacterization methods that report on the structuresnwithin these materials could clarify the effects of glycation.nFor doing this nondestructive evaluation, we employed annin situ one-photon fluorescence as well as multiphotonnmicroscopy method that combined two-photon fluores-ncence and second harmonic generation signals. We incu-nbated collagen hydrogels with glyceraldehyde, ribose, andnglucose and observed an increase in the in situ fluores-ncence and structural alterations within the materialsnduring the course of glycation. The two-photon fluores-ncence emission maximum was observed at about 460 nm.nThe fluorescence emission in the one-photon excitationnexperiment (λex 360 nm) was broad with peaksncentered at 445 and 460 nm. The 460 nm emissionncomponent subsequently became dominant during thencourse of glycation with glyceraldehyde. For the ribose,nin addition to the 460 nm peak, the 445 nm compo-nnent persisted. The glucose glycated hydrogels exhib-nited broad fluorescence that did not increase signifi-ncantly even after 6 weeks. As determined from measur-ning the fluorescence intensity at the 460 nm maximum,nglycation with glyceraldehyde was faster compared tonribose and generated stronger fluorescence signals.nUpon excitation of glycated samples with 330 nm light,ndifferent emission peaks were observed.