Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a fatal, progressive interstitial lung disease characterized by irreversible fibrosis and impaired epithelial regeneration that follows a distinct distal-to-proximal pattern. The mechanisms underlying this spatial progression of lung scarring remain unknown. Increasing evidence suggests that dysfunction and depletion of type II alveolar epithelial cells (AEC2s), the stem/progenitor cells of the alveoli, are central to disease onset and progression. However, their spatial distribution and number within the fibrotic lung remain poorly characterized, largely due to limitations of conventional two-dimensional imaging, where the sample is decontextualized from its environment and comprehensive analysis is cumbersome. Methods We developed a whole-lung optical clearing and imaging pipeline that preserves fluorescence and protein integrity, enabling high-resolution 3D visualization of intact lungs. Murine lungs were fixed, decolorized, delipidized, and then equilibrated into refraction index matching solution using a modified PEGASOS clearing protocol adapted for the lung. In addition, we generated unique transgenic mouse models to facilitate enumeration and mapping of specific cell types. We compared two high resolution imaging modalities; ribbon scanning confocal microscopy (RSCM) and mesoscale selective plane illumination microscopy (MesoSPIM) and delineated their complementary capabilities. Results Applying this approach to several lineage-labeled transgenic lines, we visualized every club cell and AEC2 in the murine lung. Moreover, structural changes were visualized in an elastase-induced injury model revealing striking regional heterogeneity in epithelial damage and alveolar remodeling, underscoring the importance of volumetric analysis in models of lung injury. Finally, we engineered a novel dual-fluorescent reporter mouse to specifically label and quantify AEC2s in3D, allowing unbiased enumeration and spatial analysis of AEC2 throughout the entire lung. Conclusions Together, this pipeline provides a powerful framework for mapping lung architecture and spatial organization in the whole lung, offering new insights into cellular and structural changes that occur during injury and aging. Figure 1. Representative images of 3D reconstructed and optical sections of airway-labeled murine lungs. Scale bars: 1000 μm (A, C) and 200 μm (B, D). This abstract is funded by: NIH/NHLBI
Peddibhotla et al. (Fri,) studied this question.