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Histological examination achieves sub-micrometer resolution laterally. In the third dimension, however, resolution is limited to section thickness. In addition, histological sectioning and mounting sections on glass slides introduce tissue-dependent stress and strain. In contrast, state-of-the-art hard X-ray micro computed tomography (μCT) systems provide isotropic sub-micrometer resolution and avoid sectioning artefacts. The drawback of μCT in the absorption contrast mode for visualising physically soft tissue is a low attenuation difference between anatomical features. In this communication, we demonstrate that formalin-fixed paraffin-embedded human cerebellum yields appropriate absorption contrast in laboratory-based μCT data, comparable to conventional histological sections. Purkinje cells, for example, are readily visible. In order to investigate the pros and cons of complementary approaches, two- and three-dimensional data were manually and automatically registered. The joint histogram of histology and the related μCT slice allows for a detailed discussion on how to integrate two-dimensional information from histology into a three-dimensional tomography dataset. This methodology is not only rewarding for the analysis of the human cerebellum, but it also has relevance for investigations of tissue biopsies and post-mortem applications. Our data indicate that laboratory-based μCT as a modality can fill the gap between synchrotron radiation-based μCT and histology for a variety of tissues. As the information from haematoxylin and eosin (H&E) stained sections and μCT data is related, one can colourise local X-ray absorption values according to the H&E stain. Hence, μCT data can correlate and virtually extend two-dimensional (2D) histology data into the third dimension.

Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.

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