Rapid chemical clearing of white matter in the post-mortem human brain by 1,2-hexanediol delipidation

Three-dimensional (3D) imaging based on chemical tissue clearing in the post-mortem human brain is a promising approach for stereoscopic understanding of central nervous system diseases. Especially, delipidation of lipid-rich white matter (WM) is a rate-determining step in human brain clearing by hydrophilic reagents. In this study, we described the rapid delipidation of WM by a 1,2-hexanediol (HxD)-based aqueous solution. HxD delipidation enabled rapid clearing of a formalin-fixed human brain specimen including the WM. Although harsh HxD delipidation was applied to the brain tissue, conventional pathological staining patterns and various types of antigenicity were sufficiently preserved. Furthermore, HxD delipidation was compatible with 3D imaging of fluorescently-labeled tissue samples. HxD delipidation could be useful in future 3D neuropathological diagnosis.     

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

Background

Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.

Methods

FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.

Results

The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.

Conclusion

We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.     

A technique for the quantification of the 3D connectivity of thin articulations in Bony sutures

The anatomy and development of cranial and facial sutures have been studied in detail using histological sections, 2D radiographs and more recently CT imaging. However, little attention has been paid to evaluating and quantifying the connectivity of these thin cortical bone articulations. More recent technological advances such as micro-CT imaging has the potential to be used to provide quantitative measurements of 3D connectivity in bony articulations. This study presents a new technique for quantifying the connectivity of bony projections inside cranial and facial sutures using a combination of skeletonization, thinning algorithms and 3D intensity mapping. The technique is demonstrated in five sutures through semi-automated analysis and image processing of μCT scans. In the sagittal, coronal and frontozygomatic sutures an average bone connectivity of 6.6–11.6% was found with multiple bony projections providing an interlocking structure between adjacent bones. Much higher bone connectivity was present in the zygomaticotemporal and zygomaticomaxillary sutures (22.7–37.4%) with few bony projections. This method combining μCT scanning and image processing techniques was successfully used to quantify the connectivity of thin bone articulations and allowed detailed assessment of sutural fusion in 3D. The wider application of this technique may allow quantification of connectivity in other structures, in particular fracture healing of long bones.     

Apports de l’imagerie hybride TEMP/TDM dans le descellement aseptique de prothèse de hanche

Hip prosthesis loosening detection is a common indication for bone scan, however, the diagnosis can be difficult to confirm based solely on bone scan information which have a low specificity. The development of bone SPECT/CT hybrid imaging allows the correlation of two complementary modalities to palliate the low sensitivity of CT caused by metallic artefacts and the moderate specificity of bone scan. It becomes a necessity for nuclear medicine physicians to master the bases of pathological hip prosthesis radiological semiology. We propose in this review article to recall the physiopathology of prosthesis loosening then we will describe the radiological and scintigraphic characteristics of hip prosthesis loosening as well as its differential diagnoses and the benefits of other imaging modalities.     

REMOVING GOLD COATING FROM SEM SAMPLES

Abstract:  An essential tool in micropalaeontological studies is analysis and imaging using a scanning electron microscope (SEM). It is commonly necessary to coat the sample with a thin layer of gold or gold-palladium alloy in order to prevent charging of the surface, to promote the emission of secondary electrons so that the specimen conducts evenly, and to provide a homogeneous surface for analysis and imaging. However, coating a specimen with gold is a semi-destructive process that masks the surface of specimens, and a common curatorial practice is to prohibit coating of important specimens. We describe a non-destructive, simple and inexpensive technique that uses KCN (or NaCN), a glass jar, paper towels, cardboard and a fume hood for the removal of gold coatings from SEM samples. This technique requires little additional handling of the specimen, and there is no need to place the specimen in a solution.     

Site-specific 3D imaging of cells and tissues with a dual beam microscope

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.     

显微CT技术在澄江生物群节肢动物研究中的应用—以Misszhouia longicaudata为例*

本研究以云南寒武纪澄江生物群节肢动物周小姐虫(Misszhouia longicaudata)为例,使用显微CT(Computed Tomography,计算机断层扫描的简写)技术对其保存在化石围岩内部的结构信息进行提取。通过Drishti软件对CT数据进行处理后,研究者可以在不破坏标本的前提下复原该动物腿肢较为完整的三维细节。此研究方法一方面对传统研究方法进行了重要补充,另一方面在研究中也对保存精美的澄江生物群化石起到保护作用,对这类标本的研究和保护具有重要意义。     

Determinants of RNA quality from FFPE samples

The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.     

Development and validation of a finite element model to simulate the opening of a medial opening wedge high tibial osteotomy

Medial opening wedge high tibial osteotomy (MOWHTO) is a surgical procedure intended to alter the coronal and sagittal plane alignment of the lower limb to primarily relieve the symptoms of osteoarthritis in the medial compartment of the knee. The purpose of this work was to develop and validate a finite element model to simulate the opening of a high tibial osteotomy and determine whether a pilot hole at the cortical hinge reduces the risk of lateral cortical fracture. Fifteen models were reconstructed from CT images of eight cadaveric specimens. The validated models indicated that the addition of the pilot hole increased the stresses and likelihood of a type-I and type-II fractures during the opening of a medial open wedge high tibial osteotomy compared to the no-hole condition.     

X-Ray Phase-Contrast CT of a Pancreatic Ductal Adenocarcinoma Mouse Model

To explore the potential of grating-based x-ray phase-contrast computed tomography (CT) for preclinical research, a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) was investigated. One ex-vivo mouse specimen was scanned with different grating-based phase-contrast CT imaging setups covering two different settings: i) high-resolution synchrotron radiation (SR) imaging and ii) dose-reduced imaging using either synchrotron radiation or a conventional x-ray tube source. These experimental settings were chosen to assess the potential of phase-contrast imaging for two different types of application: i) high-performance imaging for virtual microscopy applications and ii) biomedical imaging with increased soft-tissue contrast for in-vivo applications. For validation and as a reference, histological slicing and magnetic resonance imaging (MRI) were performed on the same mouse specimen. For each x-ray imaging setup, attenuation and phase-contrast images were compared visually with regard to contrast in general, and specifically concerning the recognizability of lesions and cancerous tissue. To quantitatively assess contrast, the contrast-to-noise ratios (CNR) of selected regions of interest (ROI) in the attenuation images and the phase images were analyzed and compared. It was found that both for virtual microscopy and for in-vivo applications, there is great potential for phase-contrast imaging: in the SR-based benchmarking data, fine details about tissue composition are accessible in the phase images and the visibility of solid tumor tissue under dose-reduced conditions is markedly superior in the phase images. The present study hence demonstrates improved diagnostic value with phase-contrast CT in a mouse model of a complex endogenous cancer, promoting the use and further development of grating-based phase-contrast CT for biomedical imaging applications.     

Searching for DNA in museum specimens: a comparison of sources in a mammal species

The number of genetic studies that use preserved specimens as sources of DNA has been steadily increasing during the last few years. Therefore, selecting the sources that are more likely to provide a suitable amount of DNA of enough quality to be amplified and at the minimum cost to the original specimen is an important step for future research. We have compared different types of tissue (hides vs. bones) from museum specimens of Iberian lynx and multiple alternative sources within each type (skin, footpad, footpad powder, claw, diaphysis, maxilloturbinal bone, mastoid process and canine) for DNA yield and probability of amplification of both mitochondrial and nuclear targets. Our results show that bone samples yield more and better DNA than hides, particularly from sources from skull, such as mastoid process and canines. However, claws offer an amplification success as high as bone sources, which makes them the preferred DNA source when no skeletal pieces have been preserved. Most importantly, these recommended sources can be sampled incurring minimal damage to the specimens while amplifying at a high success rate for both mitochondrial and microsatellite markers.     

Insights into a mummy: a paleoradiological analysis

The aim of the study was to analyze possible human skeletal remains within the wrappings of a mummy from the Archaeological Museum, Zagreb, Croatia through the use of the multidetector CT (MDCT) technology. Plain X-ray films and MDCT images of the mummy were taken in both frontal and lateral views. In a single volumetric acquisition of the whole body by MDCT 0.75 mm axial slices were obtained and combined with sagittal and coronal reformatting and three-dimensional (3D) reconstruction. Sex and age was assessed visually using standard anthropological methods. The results suggest that the mummy was of an adult female, most likely over 40 years of age at death. Pathologies observed included degenerative changes on the vertebral column and healed fractures of the lower right arm. Damage of the ethmoid bone at the roof of the nasal cavity was most likely caused by mortuary brain removal practice. Remnants of a resin and an unusual object were found inside the cranial cavity. An elongated metal object and additional three metal "belts" can be seen on the lower portion of the body. All internal organs were removed and thoracic and abdominal cavities were filled with various substances, most likely mud and pieces of linen cloth. Our results show that the MDCT is a very useful technique for assessing the human remains in archeological samples, especially in comparison to the use of plain film (X-ray), where important details are obscured and 3D imaging impossible.     

Experimental Actinobacillus pleuropneumoniae challenge in swine: Comparison of computed tomographic and radiographic findings during disease

ABSTRACT: BACKGROUND: In pigs, diseases of the respiratory tract like pleuropneumonia due to Actinobacillus pleuropneumoniae (App) infection have led to high economic losses for decades. Further research on disease pathogenesis, pathogen-host-interactions and new prophylactic and therapeutic approaches are needed. In most studies, a large number of experimental animals are required to assess lung alterations at different stages of the disease. In order to reduce the required number of animals but nevertheless gather information on the nature and extent of lung alterations in living pigs, a computed tomographic scoring system for quantifying gross pathological findings was developed. In this study, five healthy pigs served as control animals while 24 pigs were infected with App, the causative agent of pleuropneumonia in pigs, in an established model for respiratory tract disease. RESULTS: Computed tomographic (CT) findings during the course of App challenge were verified by radiological imaging, clinical, serological, gross pathology and histological examinations. Findings from clinical examinations and both CT and radiological imaging, were recorded on day 7 and day 21 after challenge. Clinical signs after experimental App challenge were indicative of acute to chronic disease. Lung CT findings of infected pigs comprised ground-glass opacities and consolidation. On day 7 and 21 the clinical scores significantly correlated with the scores of both imaging techniques. At day 21, significant correlations were found between clinical scores, CT scores and lung lesion scores. In 19 out of 22 challenged pigs the determined disease grades (not affected, slightly affected, moderately affected, severely affected) from CT and gross pathological examination were in accordance. Disease classification by radiography and gross pathology agreed in 11 out of 24 pigs. CONCLUSIONS: High-resolution, high-contrast CT examination with no overlapping of organs is superior to radiography in the assessment of pneumonic lung lesions after App challenge. The new CT scoring system allows for quantification of gross pathological lung alterations in living pigs. However, computed tomographic findings are not informative of the etiology of respiratory disease.     

Safe Corridor to Access Clivus for Endoscopic Trans-Sphenoidal Surgery: A Radiological and Anatomical Study

Purpose

Penetration of the clivus is required for surgical access of the brain stem. The endoscopic transclivus approach is a difficult procedure with high risk of injury to important neurovascular structures. We undertook a novel anatomical and radiological investigation to understand the structure of the clivus and neurovascular structures relevant to the extended trans-nasal trans-sphenoid procedure and determine a safe corridor for the penetration of the clivus.

Method

We examined the clivus region in the computed tomographic angiography (CTA) images of 220 adults, magnetic resonance (MR) images of 50 adults, and dry skull specimens of 10 adults. Multiplanar reconstruction (MPR) of the CT images was performed, and the anatomical features of the clivus were studied in the coronal, sagittal, and axial planes. The data from the images were used to determine the anatomical parameters of the clivus and neurovascular structures, such as the internal carotid artery and inferior petrosal sinus.

Results

The examination of the CTA and MR images of the enrolled subjects revealed that the thickness of the clivus helped determine the depth of the penetration, while the distance from the sagittal midline to the important neurovascular structures determined the width of the penetration. Further, data from the CTA and MR images were consistent with those retrieved from the examination of the cadaveric specimens.

Conclusion

Our findings provided certain pointers that may be useful in guiding the surgery such that inadvertent injury to vital structures is avoided and also provided supportive information for the choice of the appropriate endoscopic equipment.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.     

Spatial and temporal continuity of kangaroo rat populations shown by sequencing mitochondrial DNA from museum specimens

Summary The advent of direct sequencing via the polymerase chain reaction (PCR) has opened up the possibility of molecular studies on museum specimens. Here we analyze genetic variation in populations over time by applying PCR to DNA extracted from museum specimens sampled from populations of one species over the last 78 years. Included in this study were 43 museum specimens of the Panamint kangaroo ratDipodomys panamintinus from localities representing each of three geographically distinct subspecies. These specimens were originally collected and prepared as dried skins in 1911, 1917, or 1937. For each specimen, a 225-bp segment of the mitochondrial genome was sequenced. These mitochondrial DNA sequences were compared to those of 63 specimens collected at the same localities in 1988. The three subspecies were nearly completely distinct. Only 2 of the 106 individuals shared mitochondrial types between subspecies. For all three localities, the diversity levels were maintained between the two temporal samples. The concordance observed between the two temporally separate phylogenies supports the use of museum specimens for phylogenetic inference. This study demonstrates the accuracy and routine nature of the use of museum specimens in the analysis of mitochondrial sequence variation in natural populations and, importantly, that a temporal aspect can now be added to such studies.     

A detection method for streak artifacts and radiological noise in a non-uniform region in a CT image

By using the CT images obtained by subtracting two CT images acquired under the same conditions and slice locations, we have devised a method for detecting streak artifacts in non-uniform regions and only radiological noise components in CT images. A chest phantom was scanned using 16- and 64-multidetector row helical CT scanners with various mAs values at 120 kVp. The upper lung slice image was employed as a target image for evaluating the streak artifacts and radiological noise. One hundred parallel line segments with a length of 80 pixels were placed on the subtracted CT image, and the largest CT value in each CT value profile was employed as a feature variable of the streak artifacts; these feature variables were analyzed with the extreme value theory (Gumbel distribution). To detect only the radiological noise, all CT values contained in the 100 line profile were plotted on normal probability paper and the standard deviation was estimated from the inclination of its fitted line for the CT value plots. The two detection methods devised in this study were able to evaluate the streak artifacts and radiological noise in the CT images with high accuracy.