Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices

Khondoker M Akram; Laura L Yates; Róisín Mongey; Stephen Rothery; David C A Gaboriau; Jeremy Sanderson; Matthew Hind; Mark Griffiths; Charlotte H Dean

doi:10.21769/BioProtoc.3403

Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices

Bio Protoc. 2019 Oct 20;9(20):e3403. doi: 10.21769/BioProtoc.3403.

Authors

Khondoker M Akram¹, Laura L Yates², Róisín Mongey², Stephen Rothery^{2

3}, David C A Gaboriau^{2

3}, Jeremy Sanderson⁴, Matthew Hind^{2

5}, Mark Griffiths^{2

6}, Charlotte H Dean^{2

4}

Affiliations

¹ Department of Oncology & Metabolism, University of Sheffield, Sheffield, UK.
² National Heart and Lung Institute, Imperial College London, London, UK.
³ Facility for Imaging by Light Microscopy, NHLI, Faculty of Medicine, Imperial College London, London, UK.
⁴ MRC Harwell Institute, Harwell Campus, Oxfordshire, UK.
⁵ National Institute for Health Research (NIHR) Respiratory Biomedical Research Unit at the Royal Brompton & Harefield NHS Foundation Trust and Imperial College, London, UK.
⁶ Peri-Operative Medicine Department, St Bartholomew's Hospital, London, UK.

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.

Keywords: Deconvolution software; Imaging alveologenesis; Lung development; Precision-cut lung slices; Time-lapse imaging.