Comparative analysis of sample preparation protocols of soft biological tissues for morphometric studies using synchrotron-based X-ray microtomography

Abstract

The spread of microtomography as a tool for visualization of soft tissues has had a significant impact on a better understanding of complex biological systems. This technique allows a detailed three-dimensional quantitative view of the specimen to be obtained, correlating its morphological organization with its function, providing valuable insights on the functionality of the tissue. Regularly overlooked, but of great importance, proper sample mounting and preparation are fundamental for achieving the highest possible image quality even for the high-resolution imaging systems currently under development. Here, a quantitative analysis compares some of the most common sample-mounting strategies used for synchrotron-based X-ray microtomography of soft tissues: alcoholic-immersion, paraffin-embedding and critical-point drying. These three distinct sample-mounting strategies were performed on the same specimen in order to investigate their impact on sample morphology regardless of individual sample variation. In that sense, the alcoholic-immersion strategy, although causing less shrinkage to the tissue, proved to be the most unsuitable approach for a high-throughput high-resolution imaging experiment due to sample drifting. Also, critical-point drying may present some interesting advantages regarding image quality but is also incompatible with a high-throughput experiment. Lastly, paraffin-embedding is shown to be the most suitable strategy for current soft tissue microtomography experiments. Such detailed analysis of biological sample-mounting strategies for synchrotron-based X-ray microtomography are expected to offer valuable insights on the best approach for using this technique for 3D imaging of soft tissues and following morphometric analysis.