Label-free optical fingerprints of hyperoxia-induced lung alterations in preterm rabbits

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm neonates that carries significant long-term implications for respiratory health. Its pathogenesis is multifactorial, with oxygen toxicity and injury from mechanical ventilation being key contributing factors. While neonatal animal models exposed to hyperoxia are widely used to mimic human BPD, lung development is often assessed through classical histological analyses. Although these methods remain fundamental in preclinical study, they may require multiple staining techniques and sections to highlight key features of BPD. Here, we introduce a label-free, multi-modal imaging platform combining two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) to characterize the lung alterations induced by a 7-day hyperoxia (95% O2) exposure in preterm rabbits. Lung sections were obtained from preterm rabbit pups delivered at 28 gestational age (GA) (term 31 GA) either exposed to normoxia (21% O2) or hyperoxia (95% O2) for seven days. Lung sections were scanned with TPEF microscope at 780 nm and tissue intrinsic signals including autofluorescence intensity and lifetime, as well as SHG from collagen were simultaneously collected in the 470–570 nm, 420–460 nm and 380–410 nm ranges, respectively. BPD-relevant features were extracted from images, validated through traditional staining and immunolabelling and analyzed using an optimized pipeline. The tool reliability was tested by correlating label-free features with conventional Hematoxylin and eosin (H&E)-derived histomorphological parameters and lung functions measurements. This method simultaneously resolves key BPD-related features, such as tissue density (TD%), alveolar exudates, collagen deposition, arterial medial thickness (MT%) alterations, and alveolar simplification, capturing the differences between hyperoxia and normoxia samples without dyes or antibodies. Quantitative outputs from our label-free pipeline strongly correlate with conventional histology and lung function measurements, thus validating its robustness. This approach holds promise as a powerful tool for preclinical research, enabling simultaneous imaging and quantification of multiple pathological features with a single acquisition. This tool is envisioned to be integrated with classical histology and immunolabelling for a more comprehensive and information rich assessment of tissue alterations.