Pixel super-resolved virtual staining of label-free tissue using diffusion models

Virtual staining of tissue offers a powerful tool for transforming label-free microscopy images of unstained tissue into equivalents of histochemically stained samples. This study presents a diffusion model-based pixel super-resolution virtual staining approach utilizing a Brownian bridge process to enhance both the spatial resolution and fidelity of label-free virtual tissue staining, addressing the limitations of traditional deep learning-based methods. Our approach integrates sampling techniques into a diffusion model-based image inference process to significantly reduce the variance in the generated virtually stained images, resulting in more stable and accurate outputs. Blindly applied to lower-resolution auto-fluorescence images of label-free human lung tissue samples, the diffusion-based pixel super-resolution virtual staining model consistently outperforms conventional approaches in resolution, structural similarity and perceptual accuracy, successfully achieving a pixel super-resolution factor of 4-5×, increasing the output space-bandwidth product by 16-25-fold compared to the input label-free microscopy images. Diffusion-based pixel super-resolved virtual tissue staining not only improves resolution and image quality but also enhances the reliability of virtual staining without traditional chemical staining, offering significant potential for clinical diagnostics.