Imaging the extracellular matrix structure and remodeling in healthy, fibrotic, and emphysematous human precision-cut lung slices

Human precision-cut lung slices (hPCLS) have been used for studying extracellular matrix (ECM) remodeling in lung diseases; yet, quantitative assessment of disease-induced structural changes remains limited. We utilized multiphoton microscopy combining second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and optical coherence tomography (OCT) to characterize ECM organization in hPCLS from healthy, pulmonary fibrosis (PF), and chronic obstructive pulmonary disease (COPD) donors. We applied quantitative metrics including fractal dimension, texture correlation analysis, fiber diameter and waviness measurements to assess ECM structural alterations. PF samples exhibited significantly increased fractal dimension and elevated collagen content, while COPD samples showed reduced fractal dimension and collagen content. Collagen fiber waviness, diameter and structure from texture analysis were significantly higher in diseased samples. Tissue stiffness, as a physiological readout measured from tensile tests, was significantly higher in PF than in healthy and COPD samples. The fractal dimension and collagen content estimated from standard histology showed a high correlation with collagen content (R2 = 0.92). Alveolar airspaces from OCT and histological images had slightly higher mean diameters from healthy samples than from PF and COPD samples, with diseased tissues showing increased heterogeneity characterized by collapsed alveoli clustered around enlarged airspaces. Elastase treatment of hPCLS significantly reduced texture correlation while waviness showed a similar trend, suggesting a possible role of elastic fibers in maintaining collagen network organization. Our comprehensive image and functional stiffness analyses enable quantitative assessment of ECM remodeling and offer valuable tools for monitoring structure-function alterations in lung disease.