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Purpose: X-ray phase-contrast tomography (XPCT) is a non-destructive, three-dimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi- and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive -ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected.
Approach: Differently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample.
Results: We show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains.
Conclusion: The results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources.
