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.

A quality optimization approach to image Achilles tendon microstructure by phase-contrast enhanced synchrotron micro-tomography

“…Recent studies have shown the importance of systematically studying the effect of different types of sample preparation protocols and imaging conditions to best describe the inner structure of complex soft biological tissues 21 , 33 , 41 . Fresh frozen samples imaged in saline solution enable conditions close to native.…”

“…dehydration from the beam during imaging and sample micro-movements can reduce the image quality 33 . Fixation of the tissue can increase stability, but the images could still be affected by vibrations 41 . Samples embedded in solid media are generally more stable during imaging, but they require more invasive preparation.…”

A correlation analysis of Light Microscopy and X-ray MicroCT imaging methods applied to archaeological plant remains’ morphological attributes visualization

“…Likewise, some differences in the average size of the secretory structures were observed when measured by X-ray MicroCT or HRLM images. This effect could be linked to distortions originating in sample preparation procedures, including sample dehydration and resin embedding, meaning all quantitative comparisons between specimens should be made under the same imaging method 53 . Results obtained from both imaging methods are complementary in terms of a more exhaustive analysis of morphological characteristics of plant remains.…”

Illuminating the Brain With X-Rays: Contributions and Future Perspectives of High-Resolution Microtomography to Neuroscience

“…High z-probes are contrast agents featuring high-atomic-number (high-Z) elements which absorb x-rays effectively. Examples of these probes that have already been reported for visualizing biological microstructures include osmium (Ananda et al, 2006;Johnson et al, 2006;Litzlbauer et al, 2006;Dias et al, 2019), gold (Mizutani et al, 2007(Mizutani et al, , 2008(Mizutani et al, , 2010Li et al, 2010), silver (Mizutani et al, 2008;Parameswaran et al, 2009), iodine (de Crespigny et al, 2008;Metscher, 2009), platinum (Wang et al, 2017), mercury (Fonseca et al, 2018;Nani et al, 2020), tungsten (Metscher, 2009), and lead (Kamenz and Weidemann, 2009). The advantage of this method is the clear visualization of structures that may not be well delimited only by phase contrast.…”

“…All these approaches rule out the need for fluorescence dyes and complex sample preparation. Nonetheless, XRM is still in an initial state of development creating the necessary tools focusing not only on instrumentation but also on sample preparation and mounting protocols (Dias et al, 2019).…”