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 x 1,024 pixel) were obtained. Continuous isometric micro-CT scans (voxel size 9 microm) were acquired to reconstruct two- and three-dimensional images. Tissue samples were sectioned (8-microm 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 microm) correlated well (r = 0.97; P < 0.0001) with an agreement of -0.1 +/- 3.4 microm. 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.

An Official Research Policy Statement of the American Thoracic Society/European Respiratory Society: Standards for Quantitative Assessment of Lung Structure

“…In vivo data obtained from MDCT and magnetic resonance imaging (MRI) have been compared with postoperative or postmortem micro-CT images of excised lung specimens and to morphometry (179,195,196). In one example, excised lungs were air-inflated at a constant transpulmonary pressure while suspended in liquid nitrogen fumes (71).…”

High resolution tomographic imaging of the alveolar region of the mammalian lung

“…In contrast to TEM and SEM, Computed Tomography (CT) offers a nondestructive insight into the lung Relatively low resolution micro-Computed Tomography (µCT) enables the study of the major airways of swine [79], rat [136,137] or mice lung [115,138]. Ultrahigh resolution Synchrotron Radiation based X-ray Tomographic Microscopy (SRXTM) enables the study of the functional units of the pulmonary gas exchange area, the so-called acini with unmatched resolution and precision [19,139].…”

“…Successful visualization of the acinar region, though, was limited by the spatial resolution of standard µCT. In the past few years, however, µCT-based 3d reconstruction and morphometric analysis of the alveolar region has become possible [79,83,115], facilitated by the advent of Synchrotron Radiation Computed Tomography (SRCT). SRCT is to be distinguished from SRXTM [18,48,61].…”

“…They were able to analyze the structure of one partial acinus through formidable effort and stated that the process proved to be very difficult and time consuming. Litzlbauer et al [79] analyzed porcine lungs using µCT but suggest to favor SRXTM over 1 mm Pitch-angle [141] rotation between the two datasets. (c): Pitch and roll rotation between the two datasets.…”

“…Despite the necessity of accurate 3d structural renderings of the gas exchange regions of the lung, there have been only a handful of attempts in the past (e. g. ., [76][77][78][79][80][81][82][83]), mainly because formidable effort was required using traditional specimen preparation and imaging techniques.…”