research papers J. Synchrotron Rad. (2018). 25, 1153–1161 https://doi.org/10.1107/S1600577518005489 1153 Received 9 August 2017 Accepted 9 April 2018 Edited by I. Schlichting, Max Planck Institute for Medical Research, Germany ‡ These authors contributed equally to this work. § These authors contributed equally to this work. Keywords: virtual histology; ex vivo staining; SRmCT; synchrotron radiation. Supporting information: this article has supporting information at journals.iucr.org/s Synchrotron inline phase contrast mCT enables detailed virtual histology of embedded soft-tissue samples with and without staining Mara Saccomano, a ‡ Jonas Albers, a,b ‡ Giuliana Tromba, c Marina Dobrivojevic ´ Radmilovic ´, d Srec ´ko Gajovic ´, d Frauke Alves a,b,e § and Christian Dullin a,b,c *§ a Translational Molecular Imaging, Max Planck Institute for Experimental Medicine, Germany, b Institute for Diagnostic and Interventional Radiology, University Medical Center Go ¨ ttingen, Germany, c Elettra-Sincrotrone, Trieste, Italy, d Croatian Institute for Brain Research, University of Zagreb School of Medicine, Zagreb, Croatia, and e Clinic for Heamatology and Medical Oncology, University Medical Center Go ¨ ttingen, Germany. *Correspondence e-mail: christian.dullin@elettra.eu Synchrotron radiation micro-computed tomography (SRmCT) based virtual histology, in combination with dedicated ex vivo staining protocols and/or phase contrast, is an emerging technology that makes use of three-dimensional images to provide novel insights into the structure of tissue samples at microscopic resolution with short acquisition times of the order of minutes or seconds. However, the high radiation dose creates special demands on sample preparation and staining. As a result of the lack of specific staining in virtual histology, it can supplement but not replace classical histology. Therefore, the aim of this study was to establish and compare optimized ex vivo staining and acquisition protocols for SRmCT-based virtual histology of soft-tissue samples, which could be integrated into the standard workflow of classical histology. The high grade of coherence of synchrotron radiation allows the application of propagation-based phase contrast imaging (PBI). In this study, PBI yielded a strong increase in image quality even at lower radiation doses and consequently prevented any damage to the tissue samples or the embedding material. This work has demonstrated that the improvement in contrast-to-noise ratio by PBI enabled label-free virtual histology of soft-tissue specimens embedded in paraffin to a level of detail that exceeds that achieved with staining protocols. 1. Introduction Classical histology of tissue sections in combination with chemical staining and/or immunohistochemistry (IHC) is widely used in both preclinical research and clinical routine in order to study tissue samples at a cellular level. Over the years, a variety of tissue-specific staining methods have been estab- lished, allowing a precise delineation of different morpholo- gical structures (Kienan, 2008). However, the main limitation of traditional histology is its two-dimensional nature and the destruction of the sample during the course of the procedure. Indeed, serial sectioning can be performed to assess the sample in three dimensions, but this approach is very labor intensive and the fusion of several sections into a three- dimensional volume data set has to deal with artefacts caused by the cutting process (Ourselin et al., 2001; Handschuh et al., 2010). Alternatively, three-dimensional visualization of thicker biological samples can be obtained for instance by confocal or multiphoton microscopic techniques, but only with very low penetration depth (<1 mm), or with scanning and transmission electron microscopy, which does offer high- resolution three-dimensional imaging but only of very thin samples or close to the cutting edge. ISSN 1600-5775 # 2018 International Union of Crystallography