Quantitative assessment of collagen architecture from routine histopathological images shows concordance with Second Harmonic Generation microscopy

Collagen organization within the tumour microenvironment plays a critical role in tumour progression and has emerged as an important structural biomarker in cancer. Second Harmonic Generation (SHG) microscopy enables label-free visualization and quantitative assessment of fibrillar collagen architecture; however, its high cost, specialized instrumentation, and limited field-of-view restrict routine clinical application. In this study, we evaluated whether collagen features quantified from digitally scanned Masson–Goldner’s Trichrome-stained histopathological sections can approximate measurements obtained from SHG microscopy. Formalin-fixed paraffin-embedded breast tumour tissues, including benign and invasive ductal carcinoma (IDC) samples with varying collagen content, were analysed using SHG microscopy and whole-slide brightfield imaging. Matched regions of interest were analysed using two independent digital image analysis approaches: a conventional ImageJ-based workflow (TWOMBLI) and a machine learning–based computational pipeline. Collagen structural parameters including collagen deposition area, fibre number, and alignment metrics were quantified and compared across imaging modalities using correlation analysis. SHG signals were consistently detected from Trichrome-stained sections, confirming compatibility of SHG imaging. Quantitative comparison demonstrated significant concordance between SHG-derived collagen metrics and those obtained from digital image analysis pipelines, particularly for collagen area and fibre alignment. These findings demonstrate a proof-of-concept, that computational analysis of routine histopathological images can capture key spatial features of collagen organization comparable to SHG microscopy. Digital pathology–based collagen quantification therefore represents a potentially scalable and clinically accessible approach for assessing extracellular matrix architecture in tumour tissues.