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Barbone, Giacomo E.; Bravin, Alberto; Mittone, Alberto; Kraiger, Markus J.; Hrabe de Angelis, Martin; Bossi, Mario; Ballarini, Elisa; Rodriguez-Menendez, Virginia; Ceresa, Cecilia; Cavaletti, Guido; Coan, Paola (2020): Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation. In: Journal of Neuroscience Methods, Vol. 339, 108744
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Background: Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. New Method: Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.0(3) to 0.3(3) mu m(3), and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. Results: Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Comparison with Existing Methods: Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. Conclusions: Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.