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Barbone, G.E.* ; Bravin, A.* ; Mittone, A.* ; Kraiger, M. ; Hrabě de Angelis, M. ; Bossi, M.* ; Ballarini, E.* ; Rodriguez-Menendez, V.* ; Ceresa, C.* ; Cavaletti, G.* ; Coan, P.*

Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation.

J. Neurosci. Methods 339:108744 (2020)
Postprint DOI PMC
Open Access Green
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.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Micro-ct ; X-ray Phase-contrast ; Synchrotron Radiation ; Multiscale Neuroimaging ; Spinal Cord Imaging ; Soft-tissue Fixation; Synchrotron-radiation; Diffusion Mri; Micro-ct; Tomography; Brain; Microstructure; Connectivity; Histology; Retrieval; Model
ISSN (print) / ISBN 0165-0270
e-ISSN 1872-678X
Zeitschrift Journal of Neuroscience Methods
Quellenangaben Band: 339, Heft: , Seiten: , Artikelnummer: 108744 Supplement: ,
Verlag Elsevier
Verlagsort Radarweg 29, 1043 Nx Amsterdam, Netherlands
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Experimental Genetics (IEG)