QUANTITATIVE LABEL-FREE INFRARED MICROSCOPY OF LUNG TISSUES FROM RABBIT MODELS OF BRONCHOPULMONARY DYSPLASIA

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting extremely preterm neonates, causing significant long-term respiratory consequences. While hyperoxia-exposed preterm rabbits have been proposed as BPD models in pharmacological studies, lung alterations are conventionally assessed using classical histology and immunolabelling. Although fundamental in preclinical research, these methods often require multiple stains and tissue sections to reveal key BPD features. Additionally, the limited availability of validated antibodies for rabbits demands time-consuming optimization. This study introduces 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 parenchymal alterations induced by a 7-day hyperoxia exposure in preterm rabbits. Lung sections were obtained from preterm rabbit pups delivered at 28 days of gestational age (GA) (term 31 GA) either exposed for 7 days to normoxia (21% O2) or hyperoxia (95% O2), to mimic BPD. 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 platform’s validity was confirmed by correlating label-free features with conventional H&E-derived histomorphological parameters and lung function measurements. This method simultaneously resolves key BPD-related features, such as tissue density, alveolar exudates, increased collagen deposition, arterial medial thickening, and alveolar simplification, effectively discriminating between hyperoxia and normoxia samples without dyes or antibodies. Quantitative outputs from the label-free pipeline strongly correlate with traditional histology and lung function measurements, confirming 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 label-free acquisition. Ultimately, this platform can complementconventional histology and immunolabelling to provide a more comprehensive and detailed assessment of tissue alterations.