Formaldehyde vapour fixation enables multiscale phase-contrast imaging and histological validation of human-sized lungs.

Abstract

Accurate diagnosis and characterization of lung disease increasingly rely on advanced imaging modalities capable of resolving fine microstructural details while minimizing radiation exposure. Phase-sensitive computed tomography (CT), particularly propagation-based imaging (PBI), offers superior soft tissue contrast but has historically been limited by the lack of compatible fixation techniques that preserve lung architecture post-excision. We present an adapted formaldehyde (FA) vapour fixation protocol designed to maintain human-sized lungs in a physiologically inflated and morphologically stable state. This approach prevents collapse of the delicate air-tissue interfaces, a major barrier to high-fidelity phase-contrast imaging and histological correlation. Our method enables high-resolution, multiscale imaging from whole-organ PBI at 67 µm voxel size to localized subcellular synchrotron PBI at 650 nm voxel size on the same specimen, with preserved spatial relationships critical for accurate validation of imaging findings. In porcine models, FA vapour fixation maintained alveolar integrity and radiological contrast without compromising histological detail, while also avoiding the artifacts associated with liquid fixation. Crucially, the protocol allows regulation of inflation and fixation dynamics, addressing longstanding challenges in ex vivo lung imaging and enabling consistent specimen preparation across studies. This fixation technique supports biosafe stabilization of freshly explanted human lungs-such as those from transplant procedures creating new opportunities for translational research on pathological tissue. By bridging high-resolution radiology and histopathology, our scalable fixation protocol establishes a standardized foundation for multimodal lung imaging and offers a critical tool for advancing both fundamental lung research and clinical diagnostics.