Quantitative label-free multiphoton imaging of collagen features predicts disease severity and progression in idiopathic pulmonary fibrosis

The prognostic value of deep-tissue microstructure profiling derived from transbronchial lung cryobiopsy (TBLC) specimens in idiopathic pulmonary fibrosis (IPF) remains poorly explored. This study aims to bridge this gap by quantitatively extracting collagen features in heterogeneous pathological regions using multiphoton microscopy (MPM), and providing novel insights into the potential of collagen features as sensitive biomarkers for predicting severity and disease progression in IPF. In this prospective and observational study, a total of 82 patients with IPF undergoing TBLC and 22 healthy controls were enrolled. The differences in collagen features between the normal/mild and moderate/severe groups were compared. Multivariate logistic regression was used to identify collagen features associated with disease progression. Histopathologically, 62. 2% of the cohort exhibited a probable usual interstitial pneumonia (UIP) pattern. PERMANOVA indicated pronounced divergent collagen topographies among the five pathological regions (F = 14. 14, R2 = 0. 233, P = 0. 001). Collagen features in histologically appeared normal regions exhibited significant collagen micro-remodeling compared to healthy controls (P = 0. 01). Specifically, in advanced regions comprised of destructive regions and honeycombing regions, the anisotropy index (P < 0. 001), eccentricity (P = 0. 007), and local solidity heterogeneity (P = 0. 03) were found to be decreased in comparison to the alveolar preserved fibrotic regions, while the crosslink spacing (P = 0. 001) was expanded. In the moderate-to-severe group categorized by FVC%predicted, local orientation heterogeneity was markedly elevated (P = 0. 01), and the crosslink spacing expanded (P = 0. 008). In the moderate-to-severe group categorized by DLCO%predicted, the collagen fiber complexity network was reduced (P = 0. 002). After adjusting for GAP score and antifibrotic therapy, the elevated gaborₗ4ₜh120ₘean was independently associated with disease progression (OR = 3. 25, P = 0. 009). In contrast, increased collagen fiber independent index (OR = 0. 27, P = 0. 008) and longer collagen fiber length (OR = 0. 35, P = 0. 03) were identified as protective factors. MPM imaging successfully mapped the spatial heterogeneity of the fibrotic microenvironment in IPF by quantifying collagen signatures. The morphological and structural features of collagen fibers could serve as promising pathological biomarkers for precisely assessing the severity and progression of IPF.