Abstract
Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.
Highlights
X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology
Eosin can be used to stain cytoplasm[9], while hematein functions as a nucleus specific staining agent[10,11]. While these approaches seem to take the specificity of microCT based virtual histology to a new level, they are hindered by limited sample sizes as well as by the fact that only a few applicable protocols exist to this day
We show that the combination of label-free 3D virtual histology and classical 2D paraffin-based histology can achieve a precise overlay of microCT and histology and/or immunohistochemistry by compensating for the unavoidable non-uniform deformations that occur during the sectioning and staining process
Summary
X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. We show that the combination of label-free 3D virtual histology and classical 2D paraffin-based histology can achieve a precise overlay of microCT and histology and/or immunohistochemistry by compensating for the unavoidable non-uniform deformations that occur during the sectioning and staining process.
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