Virtual staining for 3D X-ray histology of bone implants

Three-dimensional X-ray histology techniques offer a non-invasive alternative to conventional 2D histology, enabling volumetric imaging of biological tissues without the need for physical sectioning or chemical staining. However, the inherent greyscale image contrast of X-ray tomography limits its biochemical specificity compared to traditional histological stains. Within digital pathology, deep learning-based virtual staining has demonstrated utility in simulating stained appearances from label-free optical images. In this study, we extend virtual staining to the X-ray domain by applying cross-modality image translation to generate artificially stained slices from synchrotron-radiation-based micro-CT scans. Using over 50 co-registered image pairs of micro-CT and toluidine blue-stained histology from bone-implant samples, we trained a modified CycleGAN network tailored for limited paired data. Whole slide histology images were downsampled to match the voxel size of the CT data, with on-the-fly data augmentation for patch-based training. The model incorporates pixelwise supervision and greyscale consistency terms, producing histologically realistic colour outputs while preserving high-resolution structural detail. Our method outperformed Pix2Pix and standard CycleGAN baselines across SSIM, PSNR, and LPIPS metrics. Once trained, the model can be applied to full CT volumes to generate virtually stained 3D datasets, enhancing interpretability without additional sample preparation. While features such as new bone formation were able to be reproduced, some variability in the depiction of implant degradation layers highlights the need for further training data and refinement. This work introduces virtual staining to 3D X-ray imaging and offers a scalable route for chemically informative, label-free tissue characterisation in biomedical research.