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White matter fiber tracts are revealed by XPCT in a human brain and disappear upon demyelination brain clearing. (A-B) Single XPCT slice of a human brain sample from 2 distinct but adjacent blocks. (A) Ethanol dehydration reveals white matter as hyperintense areas (arrows); (B) White matter hyperintense signal is lost after demyelination by brain clearing. Other anatomical landmarks such as large vessels are clearly seen (large blue arrowheads). Because the sample was not perfused, there is also contrast from small vessels (smaller green arrowheads); (C-D) Single XPCT slice through a rodent brain (C) without and (D) with demyelination by brain clearing: note the disappearance of white matter hyperintense signal. Clearing procedure induced brain expansion and ethanol dehydration induced brain shrinkage, hence the slight difference in sample size.
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White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enabl...
Citations
... XPBI was initially developed at large-scale synchrotron facilities but has gradually been transferred to the laboratory utilizing X-ray micro-and nanofocus tubes [24], [25], [26]. Hitherto, the functionality and applicability of XPBI have been investigated for various soft tissues [27], [28], [29], [30], [31]. Due to the maturity and commercial availability of micro-and nano-CT scanners, this technology should be readily available to clinics. ...
Histological analysis is the core of follicular thyroid carcinoma (FTC) classification. The histopathological criteria of capsular and vascular invasion define malignancy and aggressiveness of FTC. Analysis of multiple sections is cumbersome and as only a minute tissue fraction is analyzed during histopathology, under-sampling remains a problem. Application of an efficient tool for complete tissue imaging in 3D would speed-up diagnosis and increase accuracy. We show that X-ray propagation-based imaging (XPBI) of paraffin-embedded tissue blocks is a valuable complementary method for follicular thyroid carcinoma diagnosis and assessment. It enables a fast, non-destructive and accurate 3D virtual histology of the FTC resection specimen. We demonstrate that XPBI virtual slices can reliably evaluate capsular invasions. Then we discuss the accessible morphological information from XPBI and their significance for vascular invasion diagnosis. We show 3D morphological information that allow to discern vascular invasions. The results are validated by comparing XPBI images with clinically accepted histology slides revised by and under supervision of two experienced endocrine pathologists.
... A diffusion-tensor imaging (DTI)-like algorithm was applied, developed by NOVITOM. 1 The algorithm, previously described by 1 https://www.novitom.com/en/ Chourrout et al. (2022a), was developed based on image gradient analysis to detect the orientation of white-matter fibers. We selected the following input parameters, adapted to the mouse brain: gradient orientation (0.5), gradient orientation tensor (0.5) and fiber orientation tensor (5). ...
To improve our understanding of the brain microstructure, high-resolution 3D imaging is used to complement classical 2D histological assessment techniques. X-ray computed tomography allows high-resolution 3D imaging, but requires methods for enhancing contrast of soft tissues. Applying contrast-enhancing staining agents (CESAs) ameliorates the X-ray attenuating properties of soft tissue constituents and is referred to as contrast-enhanced computed tomography (CECT). Despite the large number of chemical compounds that have successfully been applied as CESAs for imaging brain, they are often toxic for the researcher, destructive for the tissue and without proper characterization of affinity mechanisms. We evaluated two sets of chemically related CESAs (organic, iodinated: Hexabrix and CA4+ and inorganic polyoxometalates: 1:2 hafnium-substituted Wells-Dawson phosphotungstate and Preyssler anion), for CECT imaging of healthy murine hemispheres. We then selected the CESA (Hexabrix) that provided the highest contrast between gray and white matter and applied it to a cuprizone-induced demyelination model. Differences in the penetration rate, effect on tissue integrity and affinity for tissue constituents have been observed for the evaluated CESAs. Cuprizone-induced demyelination could be visualized and quantified after Hexabrix staining. Four new non-toxic and non-destructive CESAs to the field of brain CECT imaging were introduced. The added value of CECT was shown by successfully applying it to a cuprizone-induced demyelination model. This research will prove to be crucial for further development of CESAs for ex vivo brain CECT and 3D histopathology.
... To address this issue, we have recently proposed to use in vivo high resolution synchrotron K-edge subtraction CT (SKES-CT) as a method of reference (Tavakoli et al., 2021). In addition, in-line phase contrast CT with synchrotron x-ray (XPCT) may complement in vivo analysis as a 3-dimensional virtual histology tool that allows to visualize cells labelled with metal-based contrast agents (Albers et al., 2018;Chourrout et al., 2022;Marinescu et al., 2013b). Fig. 4 F and 5 H show examples of XPCT in rodent models of brain injury in the context of cell tracking (author's own unpublished data). ...
The activation of phagocytic cells is a hallmark of many neurological diseases. Imaging them in their 3-dimensional cerebral environment over time is crucial to better understand their role in disease pathogenesis and to monitor their potential therapeutic effects. Phagocytic cells have the ability to internalize metal-based contrast agents both in vitro and in vivo and can thus be tracked by magnetic resonance imaging (MRI) or computed tomography (CT). In this review article, we summarize the different labelling strategies, contrast agents, and in vivo imaging modalities that can be used to monitor cells with phagocytic activity in the central nervous system using MRI and CT, with a focus on clinical applications. Metal-based nanoparticle contrast agents such as gadolinium, gold and iron are ideal candidates for these applications as they have favourable magnetic and/or radiopaque properties and can be fine-tuned for optimal uptake by phagocytic cells. However, they also come with downsides due to their potential toxicity, especially in the brain where they might accumulate. We therefore conclude our review by discussing the pitfalls, safety and potential for clinical translation of these metal-based neuroimaging techniques. Early results in patients with neuropathologies such as multiple sclerosis, stroke, trauma, cerebral aneurysm and glioblastoma are promising. If the challenges represented by safety issues are overcome, phagocytic cells imaging will be a very valuable tool for studying and understanding the inflammatory response and evaluating treatments that aim at mitigating this response in patients with neurological diseases.
... X-ray 3D imaging of Aβ plaques has been described in transgenic rodent models that develop amyloidosis for the 2 past decade, using propagation-based phase contrast (either with synchrotron radiation [5,6,7,8,9,10] or with a laboratory source [11]) or using gratingbased phase contrast (with synchrotron radiation [12,13,14,5]). These techniques generate 3D images with an isotropic resolution ranging from 1 µm to 10 µm, thus providing a so-called "virtual histology" of the excised brain [6,15,16,17,18,19] with minimal preparation (fixation, dehydration or paraffin embedding). Imaging AD samples with XPCT is particularly interesting because plaques stand out in the images without the need to add any staining agents. ...
... Complete acquisition parameters are reported in Supplemental Table 1. Human samples were imaged at the ESRF, beamline ID-19, using similar technique and parameters, which were previously reported [19]. ...
... Ring artifacts were removed using an in-house tool implemented by the company NOVITOM (France; https://www.novitom.com/en/) [19]. Samples were isolated from the background (air, glass tube and ethanol) and rotated within the AMIRA 3D software (release 2021.1, ...
Amyloid-β (Aβ) plaques from Alzheimer’s Disease (AD) can be visualized ex vivo in label-free brain samples using synchrotron X-ray phase-contrast tomography (XPCT). However, for XPCT to be useful as a screening method for amyloid pathology, it is essential to understand which factors drive the detection of Aβ plaques. The current study was designed to test the hypothesis that Aβ-related contrast in XPCT could be caused by the Aβ fibrils and/or by metals trapped in the plaques. This study probed the fibrillar and elemental compositions of Aβ plaques in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and Aβ plaque characteristics. XPCT, micro-Fourier-Transform Infrared spectroscopy and micro-X-Ray Fluorescence spectroscopy were conducted on human samples (genetic and sporadic cases) and on four transgenic rodent strains (mouse: APPPS1, ArcAβ, J20; rat: TgF344). Aβ plaques from the genetic AD patient were visible using XPCT, and had higher β–sheet content and higher metal levels than the sporadic AD patient, which remained undetected by XPCT. Aβ plaques in J20 mice and TgF344 rats appeared hyperintense on XPCT images, while they were hypointense with an hyperintense core in the case of APPPS1 and ArcAβ mice. In all four transgenic strains, β-sheet content was similar, while metal levels were highly variable: J20 (zinc and iron) and TgF344 (copper) strains showed greater metal accumulation than APPPS1 and ArcAβ mice. Hence, a positive contrast formation of Aβ plaques in XPCT images appeared driven by biometal entrapment.
Graphical Abstract
Highlights
Amyloid-β plaques in the different forms of Alzheimer’s Disease have various contrasts in X-ray phase-contrast tomography
In transgenic rodents, a core-restricted, positive contrast is driven by the level of metal entrapment within plaques
In humans, greater and more diffuse metal accumulation lead to a positive contrast in a genetic case of AD
... Over the past few decades, an increasing number of studies have demonstrated the high diagnostic potential of PC and DI [1], as compared to conventional radiology, in a wide range of pathologies and applications, including mammography [2,3], osteoarticular diseases [4,5], brain [6][7][8] and pulmonary diseases [9,10]. With the emergence of partially coherent X-ray sources twenty years ago, expectations regarding PC and DI became feasible, following which several PC and DI methods have been developed at synchrotrons. ...
Since the seminal work of Roentgen, X-ray imaging mainly uses the same physical phenomenon: the absorption of light by matter. Thanks to third-generation synchrotrons that provide a high flux of quasi-coherent X-rays, we have seen in recent years new imaging concepts such as phase contrast or dark-field imaging that were later adapted to conventional X-ray sources. These innovative imaging techniques are particularly suitable for visualizing soft matter, such as biological tissues. After a brief introduction to the physical foundations of these two techniques, we present the different experimental set-ups that are now available to produce such contrasts: propagation, analyzer-based, grating interferometry and non-interferometric methods, such as coded aperture and modulation techniques. We present a comprehensive review of their principles; associated data processing; and finally, their requirements for their transfer outside of synchrotrons. In conclusion, gratings interferometry, coded aperture and modulation techniques seem to be the best candidates for the widespread use of phase contrast and dark-field imaging on low-cost X-ray sources.
... Many versatile applications have been demonstrated for X-ray phase-sensitive methods during the last two decades, from material characterization [14][15][16][17][18] to biomedical virtual 3D histology [19][20][21][22][23][24][25][26]. Various operational mechanisms have been implemented to extract such multi-contrast images by changing the configuration of X-ray radiography and tomography systems or introducing some optical elements [27]. ...
Backgound: The composition of stones formed in the urinary tract plays an important role in their management over time. The most common imaging method for the non-invasive evaluation of urinary stones is radiography and computed tomography (CT). However, CT is not very sensitive, and cannot differentiate between all critical stone types. In this study, we propose the application, and evaluate the potential, of a multi-modal (or multi-contrast) X-ray imaging technique called speckle-based imaging (SBI) to differentiate between various types of urinary stones. Methods: Three different stone samples were extracted from animal and human urinary tracts and examined in a laboratory-based speckle tracking setup. The results were discussed based on an X-ray diffraction analysis and a comparison with X-ray microtomography and grating-based interferometry. Results: The stones were classified through compositional analysis by X-ray diffraction. The multi-contrast images obtained using the SBI method provided detailed information about the composition of various urinary stone types, and could differentiate between them. X-ray SBI could provide highly sensitive and high-resolution characterizations of different urinary stones in the radiography mode, comparable to those by grating interferometry. Conclusions: This investigation demonstrated the capability of the SBI technique for the non-invasive classification of urinary stones through radiography in a simple and cost-effective laboratory setting. This opens the possibility for further studies concerning full-field in vivo SBI for the clinical imaging of urinary stones.
... 1. optimal brain tissue preparation through dehydration in ethanol, 2. fast acquisition in three different (mono, double and triple) transgenic mouse strains displaying Aβ pathology, 3. semi-automatic segmentation of Aβ plaques inside the hippocampus using open-source tools (combination of Fiji plugins), 4. and full characterization of their 3D morphology. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables myelin mapping of the whole brain [13]. ...
... After ethanol dehydration, brain anatomy, and specifically white matter tracts, was uniquely displayed on XPCT images ( Fig. 1), as reported in Part I of this series of articles [13]. One additional formaldehyde-fixed sample of the J20 strain was scanned in PBS using the same set-up and reconstruction algorithm (Fig. S1): though some Aβ plaques were visible, surrounding brain tissue exhibited low overall signal intensity, strongly contaminated by ring artefacts. ...
While numerous transgenic mouse strains have been produced to model the formation of amyloid-β (Aβ) plaques in the brain, efficient methods for whole-brain 3D analysis of Aβ deposits have to be validated and standardized. Moreover, routine immunohistochemistry performed on brain slices precludes any shape analysis of Aβ plaques, or require complex procedures for serial acquisition and reconstruction. The present study shows how in-line (propagation-based) X-ray phase-contrast tomography (XPCT) combined with ethanol-induced brain sample dehydration enables hippocampus-wide detection and morphometric analysis of Aβ plaques. Performed in three distinct Alzheimer mouse strains, the proposed workflow identified differences in signal intensity and 3D shape parameters: 3xTg displayed a different type of Aβ plaques, with a larger volume and area, greater elongation, flatness and mean breadth, and more intense average signal than J20 and APP/PS1. As a label-free non-destructive technique, XPCT can be combined with standard immunohistochemistry. XPCT virtual histology could thus become instrumental in quantifying the 3D spreading and the morphological impact of seeding when studying prion-like properties of Aβ aggregates in animal models of Alzheimer’s disease. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables 3D myelin mapping in the whole rodent brain and in human autopsy brain tissue.
... CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables myelin mapping of the whole brain [10]. and mutant MAPT [13]. ...
... Qualitative analysis. After ethanol dehydration, brain anatomy, and specifically white matter tracts, was uniquely displayed on XPCT images (Fig. 1), as reported in Part I of this series of articles [10]. ...
While numerous transgenic mouse strains have been produced to model the formation of amyloid-β (Aβ) plaques in the brain, efficient methods for whole-brain 3D analysis of Aβ deposits are lacking. Moreover, standard immunohistochemistry performed on brain slices precludes any shape analysis of Aβ plaques. The present study shows how in-line (propagation-based) X-ray phase-contrast tomography (XPCT) combined with ethanol-induced brain sample dehydration enables hippocampus-wide detection and morphometric analysis of Aβ plaques. Performed in three distinct Alzheimer mouse strains, the proposed workflow identified differences in signal intensity and 3D shape parameters: 3xTg displayed a different type of Aβ plaques, with a larger volume and area, greater elongation, flatness and mean breadth, and more intense average signal than J20 and APP/PS1. As a label-free non-destructive technique, XPCT can be combined with standard immunohistochemistry. XPCT virtual histology could thus become instrumental in quantifying the 3D spreading and the morphological impact of seeding when studying prion-like properties of Aβ aggregates in animal models of Alzheimer’s disease. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables 3D myelin mapping in the whole rodent brain and in human autopsy brain tissue.
Highlights
X-ray phase-contrast tomography (XPCT) enables whole brain detection of Aβ plaques
Morphometric parameters of Aβ plaques may be readily retrieved from XPCT data
New shape parameters were successfully extracted from three Alzheimer’s disease models
A Fiji-based “biologist-friendly” analysis workflow is proposed and shared
XPCT is a powerful virtual histology tool that requires minimal sample preparation
Graphical abstract
The current consensus holds that optically‐cleared specimens are unsuitable for Magnetic Resonance Imaging (MRI); exhibiting absence of contrast. Prior studies combined MRI with tissue‐clearing techniques relying on the latter's ability to eliminate lipids, thereby fostering the assumption that lipids constitute the primary source of ex vivo MRI‐contrast. Nevertheless, these findings contradict an extensive body of literature that underscores the contribution of other features to contrast. Furthermore, it remains unknown whether non‐delipidating clearing methods can produce MRI‐compatible specimens or whether MRI‐contrast can be re‐established. These limitations hinder the development of multimodal MRI‐light‐microscopy (LM) imaging approaches. This study assesses the relation between MRI‐contrast, and delipidation in optically‐cleared whole brains following different tissue‐clearing approaches. It is demonstrated that uDISCO and ECi‐brains are MRI‐compatible upon tissue rehydration, despite both methods’ substantial delipidating‐nature. It is also demonstrated that, whereas Scale‐clearing preserves most lipids, Scale‐cleared brain lack MRI‐contrast. Furthermore, MRI‐contrast is restored to lipid‐free CLARITY‐brains without introducing lipids. Our results thereby dissociate between the essentiality of lipids to MRI‐contrast. A tight association is found between tissue expansion, hyperhydration and loss of MRI‐contrast. These findings then enabled us to develop a multimodal MRI‐LM‐imaging approach, opening new avenues to bridge between the micro‐ and mesoscale for biomedical research and clinical applications.
A feature issue is being presented by a team of guest editors containing papers based on contributed submissions including studies presented at Optics and the Brain, held April 24-27, 2023 as part of Optica Biophotonics Congress: Optics in the Life Sciences, in Vancouver, Canada
