Optical clearing combined with deep imaging of large biological specimen allows organ-wide visualization of cells in three dimensions (3D) to explore regenerative processes in a spatial context. Here, we investigate the dynamics of airway regeneration following toxin-mediated epithelial injury in cleared whole lung preparations by light sheet microscopy. We use a recently developed knock-in mouse strain labeling bronchiolar Club cells (Scgb1a1-mCherry) to define an optimal clearing procedure that efficiently preserves genetically encoded fluorophores. Dehydration in pH-adjusted tert-butanol followed by clearing in ethyl cinnamate maintained maximum mCherry fluorescence while preventing unfavorable background fluorescence. We apply this technique to depict the course of bronchiolar epithelial renewal from an acute injury phase to early and late recovery stages. 3D reconstructions of whole lungs demonstrate near-complete loss of secretory Club cells throughout the entire respiratory tract 3 days post naphthalene (dpn). Multiple foci of regenerating Club cells emerge at 7 dpn, predominantly at airway bifurcations and in distal terminal bronchioles—anatomical regions assumed to harbor distinct stem/progenitor cells subsets. At 21 dpn, clusters of newly formed Club cells have largely expanded, although the bronchiolar epithelial lining continues to regenerate. This study identifies regional stem cell niches as starting points for epithelial recovery, underscores the enormous regenerative capacity of the respiratory epithelium and demonstrates the power of whole lung 3D imaging for evaluating the extent of pulmonary damage and subsequent repair processes.

光学清除与大型生物样本的深度成像相结合，可以在三个维度 (3D) 中对细胞进行全器官可视化，以探索空间环境中的再生过程。在这里，我们通过光片显微镜研究了清除的全肺制剂中毒素介导的上皮损伤后气道再生的动力学。我们使用最近开发的敲入小鼠品系标记细支气管俱乐部细胞 ( Scgb1a1-mCherry) 来定义有效保留基因编码荧光团的最佳清除程序。在 pH 值调节的叔丁醇中脱水，然后在肉桂酸乙酯中澄清，可保持最大的 mCherry 荧光，同时防止不利的背景荧光。我们应用这种技术来描述从急性损伤阶段到早期和晚期恢复阶段的细支气管上皮更新过程。全肺的 3D 重建表明，在萘 (dpn) 后 3 天，整个呼吸道几乎完全丧失了分泌性俱乐部细胞。再生 Club 细胞的多个病灶在 7 dpn 出现，主要出现在气道分叉处和远端细支气管 - 假定具有不同干/祖细胞亚群的解剖区域。在 21 dpn，尽管细支气管上皮衬里继续再生，但新形成的Club细胞簇已经大大扩大。这项研究将区域干细胞壁龛确定为上皮恢复的起点，强调了呼吸上皮的巨大再生能力，并证明了全肺 3D 成像在评估肺损伤程度和后续修复过程方面的能力。