Innovative Methodology Three-dimensional imaging and morphometric analysis of alveolar tissue from microfocal X-ray-computed tomography

Litzlbauer, Horst Detlef, Christoph Neuhaeuser, Alexander Moell, Susanne Greschus, Andreas Breithecker, Folker Ernst Franke, Wolfgang Kummer, and Wigbert Stephan Rau. Threedimensional imaging and morphometric analysis of alveolar tissue from microfocal X-ray-computed tomography. Am J Physiol Lung Cell Mol Physiol 291: L535–L545, 2006. First published May 5, 2006; doi:10.1152/ajplung.00088.2005.—We evaluated microfocal X-ray-computed tomography (micro-CT) as a method to visualize lung architecture two and three dimensionally and to obtain morphometric data. Inflated porcine lungs were fixed by formaldehyde ventilation. Tissue samples (8-mm diameter, 10-mm height) were stained with osmium tetroxide, and 400 projection images (1,024 1,024 pixel) were obtained. Continuous isometric micro-CT scans (voxel size 9 m) were acquired to reconstruct twoand three-dimensional images. Tissue samples were sectioned (8m thickness) for histological analysis. Alveolar surface density and mean linear intercept were assessed by stereology-based morphometry in micro-CT scans and corresponding histological sections. Furthermore, stereologybased morphometry was compared with morphometric semi-automated micro-CT analysis within the same micro-CT scan. Agreement of methods was assessed by regression and Bland-Altman analysis. Comparing histology with micro-CT, alveolar surface densities (35.4 2.4 vs. 33.4 1.9/mm, P 0.05) showed a correlation (r 0.72; P 0.018) with an agreement of 2 1.6/mm; the mean linear intercept (135.7 14.5 vs. 135.8 15 m) correlated well (r 0.97; P 0.0001) with an agreement of 0.1 3.4 m. Semi-automated micro-CT analysis resulted in smaller alveolar surface densities (33.4 1.9 vs. 30.5 1/mm; P 0.01) with a correlation (r 0.70; P 0.023) and agreement of 2.9 1.4/mm. Non-destructive micro-CT scanning offers the advantage to visualize the spatial tissue architecture of small lung samples two and three dimensionally.