{Reference Type}: Journal Article
{Title}: Investigating biochemical and structural changes of glycated collagen using multimodal multiphoton imaging, Raman spectroscopy, and atomic force microscopy.
{Author}: Quansah E;Shaik TA;Çevik E;Wang X;Höppener C;Meyer-Zedler T;Deckert V;Schmitt M;Popp J;Krafft C;
{Journal}: Anal Bioanal Chem
{Volume}: 415
{Issue}: 25
{Year}: 2023 Oct 29
{Factor}: 4.478
{DOI}: 10.1007/s00216-023-04902-5
{Abstract}: Advanced glycation end products (AGEs) form extracellular crosslinking with collagenous proteins, which contributes to the development of diabetic complications. In this study, AGEs-related pentosidine (PENT) crosslinks-induced structural and biochemical changes are studied using multimodal multiphoton imaging, Raman spectroscopy and atomic force microscopy (AFM). Decellularized equine pericardium (EP) was glycated with four ribose concentrations ranging between 5 and 200 mM and monitored for up to 30 days. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopic imaging probed elastin and collagen fibers, respectively. The glycated EP showed a decrease in the SHG intensities associated with loss of non-centrosymmetry of collagen and an increase of TPEF intensities associated with PENT crosslinks upon glycation. TPEF signals from elastin fibers were unaffected. A three-dimensional reconstruction with SHG + TPEF z-stack images visualized the distribution of collagen and elastin within the EP volume matrix. In addition, Raman spectroscopy (RS) detected changes in collagen-related bands and discriminated glycated from untreated EP. Furthermore, AFM scans showed that the roughness increases and the D-unit structure of fibers remained unchanged during glycation. The PENT crosslinked-induced changes are discussed in the context of previous studies of glutaraldehyde- and genipin-induced crosslinking and collagenase-induced digestion of collagen. We conclude that TPEF, SHG, RS, and AFM are effective, label-free, and non-destructive methods to investigate glycated tissues, differentiate crosslinking processes, and characterize general collagen-associated and disease-related changes, in particular by their RS fingerprints.