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

Abstract

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 μm) were acquired to reconstruct two- and three-dimensional images. Tissue samples were sectioned (8-μm 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, stereology-based 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 μm) correlated well ( r = 0.97; P < 0.0001) with an agreement of −0.1 ± 3.4 μm. 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.