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Abstract
Background Chronic obstructive pulmonary disease (COPD) is a severely debilitating lung condition characterised by airway obstruction within the distal respiratory tree. Micro-CT imaging is a novel radiographic method that can generate a three-dimensional reconstruction of human lung micro-structure at resolutions approaching 1µm. This has revealed an obliteration of terminal bronchioles that may begin before patients have symptoms. A significant challenge when imaging wax-embedded COPD lung tissue is improving the contrast-to-noise for reasonable scan durations. The low contrast within the tissue can result in image analysis taking weeks to perform so addressing this issue is critical if micro-structure is to be studied in a more robust and less time-consuming fashion.
Aims The study’s aim was to compare and quantify the effects of different contrasting techniques on the ability of micro-CT to visualise small airways <2 mm in diameter and micro-vasculature in COPD human lung tissue.
Methods Samples were obtained from formalin-fixed sub-pleural lung tissue resected from a patient with moderate COPD and were incubated in 0.1% phosphotungstic acid (PTA), 25% Lugol’s iodine, 1% silver nitrate or left unstained to act as a control. Post-incubation, samples were embedded in epoxy resin or paraffin wax and then imaged with a 225kV HMX CT scanner at Southampton University with an average voxel size of 7.6µm The data was then analysed in Image J and VGI StudioMax.
Results Staining with 0.1% PTA and 25% Lugol’s iodine significantly improved x-ray contrast (p < 0.01) with most intense staining occurring in the small airways and micro-vessels. Staining with 1% silver nitrate failed to improve contrast (p = 0.110). PTA staining enabled small airway and vascular occlusions to be three-dimensionally characterised, providing reliable quantification of the micro-structure. Fast and simple image segmentation taking 10 minutes was required to effectively map out most of the branching network of small airways and micro-vessels. Visualising micro-structure in uncontrasted control samples required complex image analysis which took four hours to complete.
Conclusion PTA staining is a simple and effective technique at increasing x-ray contrast and reducing noise in COPD lung tissue. This greatly improves the level of visualisation of micro-structure in COPD tissue, providing more efficient and reliable analysis.
After segmentation. Small airways and accompanying vessels were almost entirely segmented after 0.1% PTA staining by quickly increasing the threshold applied to the sub-volume (see the higher threshold values displayed on the histogram). The blue marker shows where the airway cross section was taken (bottom left). Measuring internal circumference proved to be highly precise.