Abstract
Alveoli are the gas-exchange units of lung. The process of alveolar development, alveologenesis, is regulated by a complex network of signaling pathways that act on various cell types including alveolar type I and II epithelial cells, fibroblasts and the vascular endothelium. Dysregulated alveologenesis results in bronchopulmonary dysplasia in neonates and in adults, disrupted alveolar regeneration is associated with chronic lung diseases including COPD and pulmonary fibrosis. Therefore, visualizing alveologenesis is critical to understand lung homeostasis and for the development of effective therapies for incurable lung diseases. We have developed a technique to visualize alveologenesis in real-time using a combination of widefield microscopy and image deconvolution of precision-cut lung slices. Here, we describe this live imaging technique in step-by-step detail. This time-lapse imaging technique can be used to capture the dynamics of individual cells within tissue slices over a long time period (up to 16 h), with minimal loss of fluorescence or cell toxicity.