Investigating biochemical and structural changes of glycated collagen using multimodal multiphoton imaging, Raman spectroscopy, and atomic force microscopy

**Background:** Advanced glycation end products (AGEs) are heterogeneous chemical products formed by nonenzymatic interactions between reducing sugars and proteins, leading to intermolecular crosslinks that alter tissue structure. Elevated AGE levels are observed in aging, diabetes, Alzheimer's disease, end-stage renal failure, cataracts, and atherosclerosis. Pentosidine (PENT) is a major fluorescent AGE crosslink that causes stiffening of collagen bundles, thickened fibrils, and loss of elasticity. Conventional detection methods like high-performance liquid chromatography are time-consuming and destructive. This study aimed to use non-invasive, label-free techniques—two-photon excited fluorescence (TPEF), second harmonic generation (SHG), Raman spectroscopy, and atomic force microscopy (AFM)—to investigate biochemical and structural changes in glycated collagen. Ribose was chosen as a crosslinker due to its faster glycation rate compared to glucose or fructose.

**Methods:** Decellularized equine pericardium (EP) tissue was cut into 1×1 cm² pieces (1 mm thick). Glycation was performed using ribose solutions at 5 mM, 50 mM, 100 mM, and 200 mM in PBS with NaHCO₃ and HEPES. Tissues were incubated at 37°C for 10, 20, and 30 days; one sample remained in PBS as control. Raman spectra were acquired using a 785 nm laser (130 mW, 4 s exposure, 100 spectra per sample as 10×10 grid). Spectra were baseline corrected using extended multiplicative signal correction and normalized to the 1451 cm⁻¹ band. Partial least squares-linear discriminant analysis (PLS-LDA) with 5-fold cross-validation was performed for classification. AFM images (2.5×2.5 µm², 512×512 pixels) were recorded in intermittent contact mode; mean square roughness was calculated. Nonlinear imaging used a picosecond Ti:sapphire laser at 832 nm (76 MHz, 2–3 ps pulses). SHG (415/3 nm bandpass) and TPEF (525/45 nm bandpass) signals were detected in epi-direction. Images were acquired at 2048×2048 pixels resolution, 1.6 µs pixel dwell time, 16 frame averaging. TPEF and SHG ratios were calculated from 10 randomly selected ROIs (100×100 pixels) per image.

**Key Results:** TPEF ratios increased with ribose concentration, ranging from 0.49–0.52 for untreated EP to near 0.6 at 100 mM and 0.63 at 200 mM after 20 and 30 days. SHG ratios decreased correspondingly. Z-stack imaging (50 µm depth for untreated, 40 µm for glycated) showed collagen fibers expanded in deeper layers of glycated tissue, while elastin fibers were concentrated in superficial layers. Raman spectroscopy revealed no new PENT-specific bands; PLS-LDA achieved classification accuracies of 65% at 10 days, 82% at 20 days, and 90% at 30 days. At 100 mM and 200 mM ribose after 30 days, sensitivity and specificity approached 100%. AFM showed mean square roughness increased from 11.64 nm (sd 1.67 nm) in untreated EP to 21.78 nm (sd 1.26 nm) in 200 mM glycated EP after 30 days. D-period length remained similar: 63.8 nm for untreated vs. 64.1 nm for glycated.

**Clinical Implications:** This study demonstrates that multimodal optical and AFM techniques can non-invasively monitor glycation-induced structural changes in collagen, which are relevant to diabetic complications and aging. The increase in TPEF signal from PENT crosslinks and decrease in SHG signal provide sensitive markers for collagen crosslinking. Raman spectroscopy with PLS-LDA can classify glycated tissue with high accuracy (90% at 30 days), offering a label-free diagnostic tool. The lack of change in D-period length suggests that glycation affects surface roughness rather than fibril periodicity, distinguishing it from other crosslinkers like genipin. These methods could be applied to assess tissue glycation in diabetic patients, potentially aiding in early detection and monitoring of collagen-related pathologies.