Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

Rodgers, Griffin; Kuo, Willy; Schulz, Georg; Scheel, Mario; Migga, Alexandra; Bikis, Christos; Tanner, Christine; Kurtcuoglu, Vartan; Weitkamp, Timm; Müller, Bert

doi:10.1016/j.jneumeth.2021.109354

Cited by 21 publications

(12 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With hard X-ray microtomography it is possible to correct these deformations and obtain additional information on the cellular level. It can be performed at one of the individual preparation steps (formalin fixation, decalcification, ethanol fixation, paraffin embedding) [17,18]. Because of the challenges in simultaneous visualization of cartilage and bone [10], the imaging was performed after formalin fixation and decalcification.…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional imaging of porcine joints down to the subcellular level

Schulz

Rodgers²,

Tanner³

et al. 2022

Developments in X-Ray Tomography XIV

View full text Add to dashboard Cite

Joint tissues consist of trabecular and cortical bone as well as calcified and hyaline cartilage, which presents a challenge for hard X-ray-based visualization on the sub-cellular level due to the wide range of local X-ray absorption values. The density of the calcified tissues requires rather high photon energy, which often leads to insufficient contrast within the cartilage and impedes the visualization of individual biological cells. Decalcification of the tissues reduces the total and local X-ray absorption values and allows for selecting a lower photon energy. Further contrast enhancement can be achieved by ethanol fixation and paraffin tissue embedding. In this study, we (i) searched for an appropriate visualization method to investigate lesions generated by a laser osteotome and (ii) visualized a decalcified porcine joint after ethanol fixation and subsequent paraffin embedding using laboratory-and synchrotron radiation-based microtomography. The experiments at the ANATOMIX beamline of Synchrotron SOLEIL were performed in off-axis scan mode with a pixel size of 1.3 µm. Individual cells in all layers of the joint could be made visible and the effect of ethanol fixation and paraffin embedding demonstrated.

show abstract

Section: Resultsmentioning

confidence: 99%

Three-dimensional imaging of porcine joints down to the subcellular level

Schulz

Rodgers²,

Tanner³

et al. 2022

Developments in X-Ray Tomography XIV

View full text Add to dashboard Cite

show abstract

“…A non-destructive method such as SRμCT that allows multiple re-scans can complement histology to mitigate such processing-induced artifacts and help understand plaque histopathology. In a future study it could be of interest to perform high-resolution SRμCT on plaque tissue at different stages of the histology preparation stages identify which stage is better suited for this type of imaging, as has been done on brain tissue by Rodgers et al [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based μCT—A comparison with histology

et al. 2022

View full text Add to dashboard Cite

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRμCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRμCT and histology. Phase-contrast SRμCT was performed with two different magnifications (voxel sizes 6.5 μm and 0.65 μm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 μm high-resolution SRμCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 μm low-resolution SRμCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRμCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method’s non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

show abstract

“…This effect can hinder morphological quantification and lead to erroneous analyses. However, compared to paraffin embedding 39,40 and critical point drying 35,41 , the sample shrinkage can be mitigate using multiple ethanol baths in ascending ethanol concentrations 37 up to 70%, thus preserving the morphology of the tissue. Furthermore, the dehydration of the sample with ethanol ensures a higher contrast with µCT 37,42 or sCT 43 .…”

Section: Advantages Of the Protocol Compared To Other Methodsmentioning

confidence: 99%

Preparation of large biological samples for high-resolution, hierarchical, multi-modal imaging

Brunet

Walsh

Wagner

et al. 2022

Preprint

View full text Add to dashboard Cite

Imaging the different scales of biological tissue is essential for understanding healthy organ behavior and pathophysiological changes. X-ray micro-tomography using both laboratory (μCT) and synchrotron sources (sCT) is a promising tool to image the 3D morphology at the macro- and micro-scale of large samples, including intact human organs. Preparation of large samples for high resolution imaging techniques remains a challenge due to limitations with current methods, such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe a protocol to prepare, stabilize, and image large soft-tissue samples with X-ray microtomography. We demonstrate the protocol using intact human organs and Hierarchical Phase-Contrast Tomography (HiP-CT) imaging at the European Synchrotron Radiation Facility, but the protocol is equally applicable to a range of biological samples, including complete organisms, for both laboratory and synchrotron source tomography. Our protocol enhances the contrast of the sample, while preventing sample motion during the scan, even in case of different sample orientations. Bubbles trapped during mounting and those formed during scanning (in case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging (MRI), CT, and histological observation. We describe a protocol for sample preparation and mounting which requires 25 to 34 days for a large organ such as an intact human brain or heart. This preparation time varies depending on the composition, size, and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local pixel size of one micron using HiP-CT.

show abstract

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

Cited by 21 publications

References 52 publications

Three-dimensional imaging of porcine joints down to the subcellular level

Three-dimensional imaging of porcine joints down to the subcellular level

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based μCT—A comparison with histology

Preparation of large biological samples for high-resolution, hierarchical, multi-modal imaging

Contact Info

Product

Resources

About