Compartment-specific collagen architecture in Crohn’s disease biopsies using SHG/TPE microscopy: a pilot feasibility study

Intestinal fibrosis in Crohn’s disease (CD) is a key determinant of stricture formation, but collagen architecture is difficult to assess on routine mucosal biopsies because of limited tissue depth and the lack of quantitative tissue-based fibrosis measures. We evaluated the feasibility of applying second harmonic generation/two-photon excitation microscopy (SHG/TPE) with qFibrosis analysis to quantify collagen microarchitecture across histologic compartments in CD biopsies. Unstained formalin-fixed paraffin-embedded endoscopic biopsies from 31 samples obtained from 20 patients with CD were analyzed using SHG/TPE imaging and qFibrosis. Regions of interest were annotated by a gastrointestinal pathologist into glands/crypts, lamina propria, muscularis mucosae, and submucosa. Quantitative morphometric parameters reflecting collagen burden, fiber/string features, and architectural organization were compared across compartments. SHG/TPE imaging with qFibrosis successfully generated compartment-level collagen morphometric data from routine FFPE biopsy tissue. Collagen content (%SHG) and aggregated collagen (%Agg) increased progressively with tissue depth, with the highest levels observed in the submucosa. In contrast, superficial compartments showed higher fiber/string-based parameters, suggesting a more dispersed collagen architecture. These findings demonstrate measurable, depth-dependent differences in collagen organization across intestinal biopsy compartments, including patterns not readily appreciable on routine histology. SHG/TPE-based qFibrosis analysis is feasible for quantitative assessment of collagen microarchitecture in routine CD biopsy specimens and identifies compartment-specific, depth-dependent collagen patterns. However, because this pilot study lacked non-IBD controls and paired histochemical validation with Sirius Red or trichrome staining, these findings should not be interpreted as disease-specific or clinically validated. These preliminary findings support further validation of SHG/TPE-based collagen morphometry as a potential tissue-level approach for studying intestinal fibrosis in larger cohorts with controls, paired histochemical validation, histologic correlation, and clinical outcomes.