3D mapping of blood vessel networks and cells in COPD and non-COPD lung tissue samples using micro-computed tomography and immunofluorescence

Micro-computed tomography (µCT) is a non-destructive 3D imaging technique used to map tissue microstructure at typical resolutions of 1-10 µm. Correlated with 2D immunofluorescence (IF) microscopy pathophysiologically relevant cells can be identified in 3D. We aimed to investigate 3D networks of small blood vessels (<2mm) and the distribution of immune cells in mild-moderate COPD patients. FFPE peripheral lung samples from 5 non-COPD and 5 COPD patients were scanned using µCT. IF staining for smooth muscle actin, airway epithelium, mast cells and macrophages was digitised, and co-registered with 3D µCT scans. Blood vessels, airways and infiltrating cells were identified semi-automatically by IF in the µCT volume (Fig 1). Quantitative estimates of blood vessel thickness were made, initial modal thickness values were lower in non-COPD (12-25µm) compared to COPD samples (40-60µm). Thousands of macrophages (2100-7200) and mast cells (1700-9000) were localised by IF per tissue section with fewer mast cells (<1500) detected in COPD tissue sections. Combined with the 3D vasculature network the distribution of macrophages and mast cells were found to be similar in both COPD and non-COPD with 70-80% of cells within 2mm of a blood vessel. In summary, we demonstrate an approach to identify, localise and analyse lung networks in 3D and relevant infiltrating cells in human lung disease.