1. Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation
- Author
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Giacomo E. Barbone, Elisa Ballarini, Martin Hrabě de Angelis, M Bossi, Markus J. Kraiger, Alberto Mittone, Alberto Bravin, Cecilia Ceresa, Virginia Rodriguez-Menendez, Paola Coan, Guido Cavaletti, Barbone, G, Bravin, A, Mittone, A, Kraiger, M, Hrabe de Angelis, M, Bossi, M, Ballarini, E, Rodriguez-Menendez, V, Ceresa, C, Cavaletti, G, and Coan, P
- Subjects
Micro-CT ,X-ray phase-contrast ,0301 basic medicine ,Materials science ,Rodentia ,computer.software_genre ,03 medical and health sciences ,chemistry.chemical_compound ,Percutaneous Coronary Intervention ,0302 clinical medicine ,Neuroimaging ,Voxel ,medicine ,Fluorescence microscope ,Animals ,Image resolution ,Aldehydes ,Synchrotron radiation ,medicine.diagnostic_test ,X-Rays ,Soft-tissue fixation ,General Neuroscience ,Spinal cord imaging ,Magnetic resonance imaging ,X-Ray Microtomography ,Osmium ,Spinal cord ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Spinal Cord ,Multiscale neuroimaging ,chemistry ,Osmium tetroxide ,Glutaraldehyde ,computer ,030217 neurology & neurosurgery ,Biomedical engineering - Abstract
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 3.03 to 0.33 μm3, 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 9.4 T 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.
- Published
- 2020