Soft Tissue Staining Research Articles

Scaling up contrast-enhanced micro-CT imaging: Optimizing contrast and acquisition for large ex-vivo human samples

Microfocus Computed Tomography (Micro-CT) is a novel method for non-destructive 3D imaging of samples, reaching microscale resolutions. While initially prominent in material sciences for small samples, micro-CT now gains significance in biological and medical studies. Here we present our utilization of micro-CT for imaging large ex-vivo human samples for anatomical and forensic research in three recent experiments and discuss the fundamentals of micro-CT imaging.For pelvic anatomical research, whole human pelvises were imaged to explore nerve anatomy around the prostate using various concentrations of buffered lugol (B-lugol). Advanced acquisition protocols were essential due to X-ray attenuation properties of the sample, which required higher energy for sufficient photon transmission.For fetal research, B-lugol stained fetuses of 20–24 gestational weeks underwent full body imaging. However, this led to challenging acquisition parameters and images of insufficient quality. Subsequent destaining yielded less dense, yet contrast-maintaining samples allowing higher quality images. Refined acquisition protocols with reduced energy improved image quality.For forensic research, explanted hyoid-larynx complexes were imaged. Micro-CT imaging showed potential in visualizing micro-fractures. The addition of B-lugol allowed for excellent soft tissue contrast and promising possibilities for forensic evaluation.In conclusion, micro-CT imaging accommodates a diversity of large ex-vivo human samples for anatomical and forensic purposes, though challenges arise with optimal soft tissue staining and acquisition protocols. We describe partial destaining as a new possibility to alleviate scanning issues to improve scan quality and highlight topics for future research. Micro-CT imaging is a promising new avenue for medical research and forensic evaluation.

Exploring a safer alternative to eosin in soft tissue staining.

Hematoxylin-eosin (H&E) stain has stood the test of time as the standard stain for histologic examination of human tissues. Haematoxylin is a natural dye, on the contrary, its counterstain eosin is a synthetic dye which belongs to the xanthene group. Synthetic dyes are hazardous to human and animal health. With the increasing awareness of a green earth, it is advisable to use environment-friendly and biodegradable materials. Therefore, an attempt was made to develop as biofriendly substitute in the form of food colour as a counterstain for haematoxylin. To assess the staining ability of food colouring agents in routine staining and to compare its staining efficacy with Eosin. Two food colours were obtained and stain was prepared by using 70% ethyl alcohol as counterstain for haematoxylin. Different tissue structures such as epithelium, keratin, collagen fibers, muscles, salivary glands, adipocytes, blood vessels, RBCs were observed and evaluated. Group A -10 slides stained with green food colour, Group B - 10 slides stained with tomato red food colour and Group C - 10 slides stained with conventional H and E. The stained sections were assessed and graded for nuclear staining, cytoplasmic staining, clarity, uniformity and crispness of staining. The non-parametric Kruskal-Wallis and Mann-Whitney U tests were performed for statistical analysis. There was no statistically significant difference between the three study groups with respect to all the parameters except crispness of staining. The crispness of Tomato Red and H and E was better compared to green food colour. Food colouring agents can be used as a safe, biofriendly and inexpensive substitute to eosin in conventional soft tissue staining.

High Resolution Imaging of Mouse Embryos and Neonates with X-Ray Micro-Computed Tomography.

Iodine-contrast micro-computed tomography (microCT) 3D imaging provides a non-destructive and high-throughput platform for studying mouse embryo and neonate development. Here we provide protocols on preparing mouse embryos and neonates between embryonic day 8.5 (E8.5) to postnatal day 4 (P4) for iodine-contrast microCT imaging. With the implementation of the STABILITY method to create a polymer-tissue hybrid structure, we have demonstrated that not only is soft tissue shrinkage minimized but also the minimum required time for soft tissue staining with iodine is decreased, especially for E18.5 to P4 samples. In addition, we also provide a protocol on using commercially available X-CLARITYTM hydrogel solution to create the similar polymer-tissue hybrid structure on delicate early post-implantation stage (E8.5 to E14.5) embryos. With its simple sample staining and mounting processes, this protocol is easy to adopt and implement for most of the commercially available, stand-alone microCT systems in order to study mouse development between early post-implantation to early postnatal stages. © 2019 by John Wiley & Sons, Inc.

3D Anatomy and Muscle Architecture of the Human Hand: new approaches for imaging and education

New three‐dimensional digital imaging technologies permit much richer reconstructions of soft‐tissue anatomy. We used algorithmic fiber‐tracking software, or 3D muscle tractography to model the muscle architecture of distal forearm and hand muscles via diffusible iodine contrast‐enhanced computed tomography (DiceCT) of a deidentified donor. Excellent soft‐tissue staining and high CT resolution (<50 μm) allowed for the visualization of many tissues difficult to simultaneously resolve using other imaging modalities, including muscle fascicles, integumentary collagen fibers, as well as bony trabeculae. Muscle fascicles were tracked and modeled as curved cylinders from which fascicle‐specific information, such as length and orientation, was gathered. By modeling numerous fascicles within a whole muscle, we collected muscle‐wide distributions, rather than single‐value estimates, of quantitative muscle architecture variables. These comprehensive data afforded by DiceCT and algorithmic fascicle modeling promise to aid in biomechanical modeling. Further, the volume and fascicle models produced by these methods are an excellent teaching resource in medical education.Support or Funding InformationNSF IOS 1457319, NSF EAR 1631684, Missouri Research Council, Life Sciences Research Fellowship, University of Missouri Department of Pathology and Anatomical SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens).

Starfish (order: Asteroidea) possess a complex endoskeleton composed of thousands of calcareous ossicles. These ossicles are embedded in a body wall mostly consisting of a complex collagen fiber array. The combination of soft and hard tissue provides a challenge for detailed morphological and histological studies. As a consequence, very little is known about the general biomechanics of echinoderm endoskeletons and the possible role of ossicle shape in enabling or limiting skeletal movements. In this study, we used high-resolution X-ray microscopy to investigate individual ossicle shape in unprecedented detail. Our results show the variation of ossicle shape within ossicles of marginal, reticular and carinal type. Based on these results we propose an additional classification to categorize ossicles not only by shape but also by function into 'connecting' and 'node' ossicles. We also used soft tissue staining with phosphotungstic acid successfully and were able to visualize the ossicle ultrastructure at 2-μm resolution. We also identified two new joint types in the aboral skeleton (groove-on-groove joint) and between adambulacral ossicles (ball-and-socket joint). To demonstrate the possibilities of micro-computed tomographic methods in analyzing the biomechanics of echinoderm skeletons we exemplarily quantified changes in ossicle orientation for a bent ray for ambulacral ossicles. This study provides a first step for future biomechanical studies focusing on the interaction of ossicles and soft tissues during ray movements.

Bismuth-Oxo-Clusters for Soft-Tissue Staining

Bismuth-Oxo-Clusters for Soft-Tissue Staining

Comparison of decalcifying agents and techniques for human dental tissues

Teeth are among the hardest animal tissues, because they are composed of large amounts of inorganic compounds. Consequently, teeth are difficult to prepare for microscopic examination. Acids and chelating agents traditionally have been used to remove calcium ions. We compared decalcifying agents including strong acids, weak acids, chelating agents, techniques using electric current, agitation and heat. Freshly extracted teeth were fixed and decalcified using formic acid-formalin, formal-nitric acid, formalin-EDTA, Von Ebner’s fluid and Perenyi’s fluid. Three additional techniques including formic acid with agitation, formic acid with heat and formic acid with electric current also were evaluated. Decalcified teeth were evaluated histologically for tissue preservation and staining characteristics. Formic acid with gentle agitation produced the best decalcification overall based on time required for decalcification, ease of sectioning, hard and soft tissue staining and tissue preservation. Our findings support the use of agitation with formic acid decalcification, because it reduces significantly both the time required and the deleterious effects of prolonged immersion.

Dual-energy micro-CT for quantifying the time-course and staining characteristics of ex-vivo animal organs treated with iodine- and gadolinium-based contrast agents

Chemical staining of soft-tissues can be used as a strategy to increase their low inherent contrast in X-ray absorption micro-computed tomography (micro-CT), allowing to obtain fast three-dimensional structural information of animal organs. Though some staining agents are commonly used in this context, little is known about the staining agents’ ability to stain specific types of tissues; the times necessary to provide a sufficient contrast; and the effect of staining solution in distorting the tissue. Here we contribute to studies of animal organs (mouse heart and lungs) using staining combined with dual-energy micro-CT (DECT). DECT was used in order to obtain an additional quantitative measure for the amount of staining agents within the sample in 3D maps. Our results show that the two staining solutions used in this work diffuse differently in the tissues studied, the staining times of some tens of minutes already produce high-quality micro-CT images and, at the concentrations applied in this work, the staining solutions tested do not cause relevant tissue distortions. While one staining solution provides images of the general morphology of the organs, the other reveals organs’ features in the order of a hundred micrometers.

Sine systemate chaos? A versatile tool for earthworm taxonomy: non-destructive imaging of freshly fixed and museum specimens using micro-computed tomography.

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (μCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that μCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens.

BackgroundAlthough molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.FindingsScout images revealed full penetration of tissues by the staining agent. The attained isotropic voxel resolutions permit identification of internal and external structures conventionally used in earthworm taxonomy. The μCT projection and reconstruction images have been deposited in the online data repository GigaDB and are publicly available for download.ConclusionsThe dataset presented here shows that earthworms constitute suitable candidates for μCT scanning in combination with soft tissue staining. Not only are the data comparable to results derived from traditional dissection techniques, but due to their digital nature the data also permit computer-based interactive exploration of earthworm morphology and anatomy. The approach pursued here can be applied to freshly fixed as well as museum specimens, which is of particular importance when considering the use of rare or valuable material. Finally, a number of aspects related to the deposition of digital morphological data are briefly discussed.

Synchrotron- and laboratory-based X-ray phase-contrast imaging for imaging mouse articular cartilage in the absence of radiopaque contrast agents

The mouse model of osteoarthritis (OA) has been recognized as the most promising research tool for the identification of new OA therapeutic targets. However, this model is currently limited by poor throughput, dependent on the extremely time-consuming histopathology assessment of the articular cartilage (AC). We have recently shown that AC in the rat tibia can be imaged both in air and in saline solution using a laboratory system based on coded-aperture X-ray phase-contrast imaging (CAXPCi). Here, we explore ways to extend the methodology for imaging the much thinner AC of the mouse, by means of gold-standard synchrotron-based phase-contrast methods. Specifically, we have used analyser-based phase-contrast micro-computed tomography (micro-CT) for its high sensitivity to faint phase changes, coupled with a high-resolution (4.5 μm pixel) detector. Healthy, diseased (four weeks post induction of OA) and artificially damaged mouse AC was imaged at the Elettra synchrotron in Trieste, Italy, using the above method. For validation, we used conventional micro-CT combined with radiopaque soft-tissue staining and standard histomorphometry. We show that mouse cartilage can be visualized correctly by means of the synchrotron method. This suggests that: (i) further developments of the laboratory-based CAXPCi system, especially in terms of pushing the resolution limits, might have the potential to resolve mouse AC ex vivo and (ii) additional improvements may lead to a new generation of CAXPCi micro-CT scanners which could be used for in vivo longitudinal pre-clinical imaging of soft tissue at resolutions impossible to achieve by current MRI technology.

Severe Metallosis Owing to Intraprosthetic Dislocation in a Failed Dual-Mobility Cup Primary Total Hip Arthroplasty

We report a case of extensive metallosis owing to an intraprosthetic dislocation of a dual-mobility cup after a primary total hip arthroplasty. A 70-year-old man was referred to us from another center with a painful right hip 3 years after the arthroplasty. Initial investigations were suspicious of osteolysis secondary to metallosis with the characteristic “bubble sign” visualized on plain radiographs. At the revision procedure, widespread black staining of soft tissues and bone was noted. The polyethylene liner had dislodged leading to erosion of the metal socket by the prosthetic head. Histopathology examination of periprosthetic tissue confirmed metallosis. To our knowledge, this is the first reported case of severe metallosis owing to a known complication of dual-mobility sockets.

Abstract No. 225 EE: DSA – CT Correlation in Patients with Hemoptysis Requiring Embolization

Bronchial arteriography in patients with hemoptysis can show hypervascularity, fistula, pseudoaneurysm, dense soft tissue staining, and vascular abnormalities, in addition to active extravasation. The importance of knowing the status of the central pulmonary artery in the setting of pulmonary-bronchial artery fistula is underestimated. This exhibit presents a pictorial review of selected cases showing relevant abnormal findings, which are important to make the decision if a bronchial artery can be embolized safely.

Bronchial Artery Embolization: Experience With 54 Patients

To report our experience with bronchial arteriography and bronchial artery embolization (BAE). A review of clinical experience to evaluate the demographics, clinical presentation, radiographic studies, bronchoscopy, and complications of bronchial arteriography and BAE at Mayo Medical Center, Rochester, MN, from 1981 to 2000. Fifty-four patients underwent bronchial arteriography. There were 34 men and 20 women with a mean age of 53 years. Hemoptysis was the most common indication in 53 patients (98%). Hemoptysis was caused by bronchiectasis (9 patients), pulmonary hypertension (9 patients), malignancy (7 patients), mycetoma (7 patients), and other identified causes (14 patients). The cause could not be identified in eight patients. Bronchoscopy was performed in 49 patients (92%), and the results identified the bleeding lobe in 32 patients, lateralized the side of the bleeding in 5 patients, and were not helpful in 12 patients. Bronchial arteriography revealed hypervascularity (45 patients), bronchial artery hypertrophy (17 patients), hypervascularity with shunting (15 patients), dense soft tissue staining (8 patients), vascular abnormalities (7 patients), and extravasation of contrast (1 patient). BAE was attempted in 54 patients, completed in 51 patients, and was unsuccessful in 3 patients. Overall, 72 embolization sessions were performed with a total of 131 arteries embolized, and the average number of arteries embolized per patient was 2.5. Control of hemoptysis was observed in 46 patients (85%) at 1 month. Rebleeding occurred within 30 days in five patients. Eight patients had recurrent hemoptysis that occurred 30 days after the procedure. The complications of embolization included subintimal dissection of a bronchial artery (two patients), bronchial arterial perforation by a guidewire (one patient), and the reflux of embolic material into the aorta without adverse sequelae (one patient). BAE is a useful therapy to control both acute and chronic hemoptysis. BAE may help to avoid surgery in patients who are not good surgical candidates. Should hemoptysis recur in these patients, repeat embolization can be performed safely.

Variation of the Holmes Method for Histologic Staining of Bone Canaliculi

AbstractThe Holmes method for soft tissue staining of nerve fibers was modified to produce excellent detail of the canalicular network in decalcified bone tissue. With simple changes in timing and the meticulous use of fresh reagents, we were able to stain bone lacunae and canaliculi in a variety of animal and human tissues. This technique produced more uniform and accurate results than the thionin/picric acid stains used previously for visualization of osteocytes. The morphologic results can be used for diagnostic purposes as well as stereological studies where the osteocyte network is of importance. (The J Histotechnol 16:355, 1993)

Histochemical localization of alkaline pyrophosphate‐ phosphohydrolase in tooth‐forming cells of rat

The activity of a pyrophosphate-splitting tissue factor in jaws, teeth and intestinal mucosa has been studied by means of histochemistry. Freeze-cut, unfixed sections of whole animals were incubated in a buffered medium (pH 8.6) containing inorganic pyrophosphate (PP) Pb2+, Mg2+, and Zn2+ at various concentrations. The effects of compound R 8231, heat, aldehyde fixation, and demineralization with EDTA were also investigated. In sections showing optimal staining, deposition of incubation products was found in the stratum intermedium and the subodontoblastic cells of the developing tooth, in the osteoblastic layers, and at the surface of the intestinal mucosa. The hard tissues were also stained except in the demineralized sections. Treatment with heat or compound R 8231 resulted in loss of originally observed soft tissue staining while short-time demineralization with EDTA enhanced the staining reaction. It is argued that a nonspecific deposition of the capturing ion, Pb2+, can hardly explain the observed soft tissue staining. The results point to the presence of a PP-splitting enzyme, and it is suggested that the enzyme exhibits features of an alkaline phosphatase with PP-phospholhydrolytic properties rather than of an inorganic pyrophosphatase.