New staining tools and developments for 3D soft tissue CT imaging

The application of 3D X-ray imaging for biological samples (e.g. biopsies) to gain a deeper understanding of microscopic structures on a (sub)cellular level is restricted by the weak attenuation contrast of soft tissue. The development of novel staining tools for X-ray soft-tissue imaging will overcome these challenges. Here, we present the application of a recently developed method combining a laboratory-based nanoscopic X-ray CT setup enabling resolutions down to 100 nm with a target-specific X-ray staining protocol. The results clearly show that the X-ray attenuation contrast in the samples is remarkably improved by our staining method and detailed tissue (sub)structures are apparent, which cannot be visualized without the staining. The nanoscopic CT data reproduce the tissue morphology with a similar level of detail as the corresponding histological light microscopy images in 2D and enable pathological characterization of the crucial structures. Beyond that, the applied method allows for visualization of the 3D tissue architecture, offering deeper insights into the 3D microscopic structure of soft-tissue. Moreover, we demonstrate the compatibility of the X-ray stain with standard histological staining methods. Beside medical research, the methodology has the potential to contribute to advances in zoology and developmental biology.