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Hollow organs such as the lungs pose a considerable challenge for post-mortem imaging in preclinical research owing to their extremely low contrast and high structural complexity. The aim of our study was to enhance the contrast of tuberculosis lesions for their stratification by 3D x-ray&-based virtual slicing. Organ samples were taken from five control and five tuberculosis-infected mice. Micro-Computed Tomography (CT) scans of the subjects were acquired in vivo (without contrast agent) and post-mortem (with contrast agent). The proposed contrast-enhancing technique consists of x-ray contrast agent uptake (silver nitrate and iodine) by immersion. To create the histology ground-truth, the CT scan of the paraffin block guided the sectioning towards specific planes of interest. The digitalized histological slides reveal the presence, extent, and appearance of the contrast agents in lung structures and organized aggregates of immune cells. These findings correlate with the contrast-enhanced micro-CT slice. The abnormal densities in the lungs due to tuberculosis disease are concentrated in the right tail of the lung intensity histograms. The increase in the width of the right tail (~376%) indicates a contrast enhancement of the details of the abnormal densities. Postmortem contrast agents enhance the x-ray attenuation in tuberculosis lesions to allow 3D visualization by polychromatic x-ray CT, providing an advantageous tool for virtual slicing of whole lungs. The proposed contrast-enhancing technique combined with computational methods and the diverse micro-CT modalities will open the doors to the stratification of lesion types associated with infectious diseases. The research leading to these results received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement no. 115337, whose resources comprise funding from the European Union Seventh Framework Programme (FP7/2007–2013) and EFPIA companies in kind contribution. This work was partially funded by projects RTC-2015-3772-1, TEC2015-73064-EXP and TEC2016-78052-R from the Spanish Ministry of Economy, TOPUS S2013/MIT-3024 project from the regional government of Madrid and by the Department of Health, UK. This study (was supported by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013–2016) and co-financed by the European Social Fund (ESF) “ESF investing in your future”. The authors thank Dr.Guembe from CIMA-Universidad de Navarra for preparing and staining the tissue sections, and to Dr. Guerrero-Aspizua and Prof. Conti of the Department of Bioengineering, Universidad Carlos III de Madrid for the pathology evaluation.