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Differential diagnosis of intracerebral hemorrhage due to traumatic and non-traumatic origin is a challenging issue, especially in the absence of visible body injuries and other signs of violence. For a forensic expert-practitioner, the main thing is objectivity, accuracy, and speed of obtaining the result, which could fully satisfy the methods of laser polarimetry in the case of differential diagnosis of hemorrhages of traumatic and non-traumatic origin in the human brain matter. The purpose: To develop forensic criteria for the differential diagnosis of intracerebral hemorrhage of traumatic and non-traumatic origin using Mueller-matrix microscopy of linear dichroism. Materials and methods: The object of the study was brain matter samples, collected in 115 corpses of both sexes aged 22 to 86 years with accurately known causes of death. The causes of death included coronary artery disease, traumatic brain hemorrhage, ischemic brain infarction, and non-traumatic brain hemorrhages. Brain matter samples have been frozen rapidly, and histological sections have been made using a freezing microtome. The slices have been evaluated using the method of azimuthal-invariant Mueller-matrix microscopy, followed by mathematical and statistical processing of the results. Results: significant topographic heterogeneity of Mueller-matrix invariants of brain matter samples linear dichroism maps among all research groups have been found. Individual and significant variations of dispersion, asymmetry, and kurtosis magnitude have confirmed the heterogeneity of Mueller-matrix invariants. The difference of images and histograms among groups is explained by the presence of necrotic changes in the case of ischemic brain necrosis and blood cells in the brain matter samples. For the set of central statistical moments of the 1st, 3rd, and 4th orders, which characterize the distributions of circular dichroism, the method of MM-microscopy in the differentiation of samples of non-hemorrhage and hemorrhage groups reaches a satisfactory level - 78% - 84%. Conclusions: Brain matter samples Muller-matrix microscopy of linear dichroism is effective for solving diagnostic problems of forensic medicine related to evaluating the cause of death from intracerebral hemorrhage of various origins. Diagnostic efficiency of Mueller-matrix mapping of polarization manifestations of linear dichroism method for intergroup differentiation of samples of deaths from traumatic hemorrhage (Group 2) and ischemic cerebral infarction (Group 3) reaches a satisfactory level of 79 - 84%.

PURPOSE Precise correlation between three-dimensional (3D) imaging and histology can aid biomechanical modeling of the breast. We develop a framework to register ex vivo images to histology using a novel cryo-fluorescence tomography (CFT) device. METHODS A formalin-fixed cadaveric breast specimen, including chest wall, was subjected to high-resolution magnetic resonance (MR) imaging. The specimen was then frozen and embedded in an optimal cutting temperature (OCT) compound. The OCT block was placed in a CFT device with an overhead camera and 50 μm thick slices were successively shaved off the block. After each shaving, the block-face was photographed. At select locations including connective/adipose tissue, muscle, skin, and fibroglandular tissue, 20 μm sections were transferred onto cryogenic tape for manual hematoxylin and eosin staining, histological assessment, and image capture. A 3D white-light image was automatically reconstructed from the photographs by aligning fiducial markers embedded in the OCT block. The 3D MR image, 3D white-light image, and photomicrographs were rigidly registered. Target registration errors (TREs) were computed based on 10 pairs of points marked at fibroglandular intersections. The overall MR-histology registration was used to compare the MR intensities at tissue extraction sites with a one-way analysis of variance. RESULTS The MR image to CFT-captured white-light image registration achieved a mean TRE of 0.73 ± 0.25 mm (less than the 1 mm MR slice resolution). The block-face white-light image and block-face photomicrograph registration showed visually indistinguishable alignment of anatomical structures and tissue boundaries. The MR intensities at the four tissue sites identified from histology differed significantly (p

Objective: The aim of this study is to evaluate histologically the healing potential of injectable macro-porous Calcium Phosphate Cement and autogenous bone. (Normal bone healing) on surgically induced bone defect on femur bone of rabbit. Materials and Methods: Twelve adult white male New Zealand rabbits were selected for this study. Rabbits were divided into two groups and the two groups according to a two evaluation periods divided into 2 subgroups. Rabbits were anesthetized with intramuscular anethesia, and two osseous defects had been created in the distal aspect of the femur bone.one of this defects injected by a grafting material (SI-HPMC CPC) and the other left empty as control (normal healing).Animal were sacrificed after two sacrifice dates and the femur bone excised for histological and statistical evaluation.esults: Rabbits from each group were sacrificed after 2 and 4 weeks. The femur bone containing the induced defect was dissected. Each femur bone was excised using hard tissue microtome. Each slice was then fixed in 10% neutral buffered formalin. The formalin-fixed bone samples were decalcified in 15% buffered formic acid solution and processed for routine histological examination using hematoxylin and eosin stain under light microscope.. Conclusion: According to our results in the present study, we can consider Si-HPMC CPC a viable alternative to the autogenous bone in the healing of osseous bone defects . In the present study, the new and simple method to prepare macroporous CPCs using Si-HPMC a foaming agent in connected syringe , result

Aim: The aim of the study is to evaluate the inhibitory effect of single-bottle and two-bottle self-etch adhesive system after surface pretreatment with 6.5% proanthocyanidin (PA) on cysteine cathepsins (CCs) present in dentin hybrid layer using gelatin zymography. Materials and Methods: Thirty-five samples of dentin measuring 3 mm × 3 mm were obtained from 35 caries-free human third molars which were divided into seven groups. The bonded specimens were cut vertically into 1 mm thick adhesive/dentin interfaces with microtome and were pulverized into powder and subjected to zymography analysis for enzymatic activity. Results: Group VI and VII which were positive control groups had shown thicker clear bands of 50 kDa. Group IV and V which had the administration of chlorhexidine showed thicker clear bands of 50 kDa which indicated slight activity of CCs. Group II in which PA used along with single-bottle system also showed thicker clear band of 59 kDa. CCs could be active due to strong acidic nature of monomer in this group. Group III in which PA used along with two bottle system had no band formation where there is crosslinking of catalytic and allosteric domains of enzymes by PA. Conclusion: Under the limitations of this study, PA proved as a non-specific natural inhibitor of CCs. Two-bottle system showed inhibition of CCs and single bottle system failed to inhibit their activity. Future research have to be done on the bond strength and durability of self-etch adhesives after using PA and its effect on long term survival of the restorations.

Aim of the study: Using the example of a clinical case, to show the importance of dynamic observation by means of various diagnostic methods of intestinal imaging, in combination with a pathomorphological confirmation method for setting the correct clinical diagnosis. Materials and methods: The studies were performed on multislice computed tomographs Brilliance CT and ICT (Philips Medical Systems) and magnetic resonance imaging machines (GE), with intravenous contrast enhancement. We used special methods of contrasting the lumen of the intestine with gradual and retrograde filling it with water. The surgical material fixed in 10% neutral formuline for 10-24 hours. Tissue fragments were processed in a Leica TP1020 histoprocessor (Leica BioSystems, Germany) according to a standard protocol, after which it was embedded in paraffin blocks. Histological sections with a thickness of 3-5 μm were made by a Leica RM2245 microtome (Leica BioSystems, Germany). The prepared histological preparations were stained with hematoxylin and eosin in a Leica Autosteiner XL histostiner (Leica BioSystems, Germany). Results: From 2013 to 2020 patient K. underwent 4 colonoscopies, 5 esophagogastroduodenoscopies, 1 Х-ray examination with small bowel series, 3 MRI studies and 5 CT studies, including those with intravenous contrast enhancement, with a total radiation exposure of 187 mSv. The diagnosis was revised from ulcerative colitis, Crohn’s disease to NC. The final diagnosis was made by the pathologist’s analysis of materials. Conclusion: The clinical case presented by us clearly demonstrates the importance of dynamic observation by computed and magnetic resonance imaging, in combination with a pathomorphological confirmation method.

Abdominal sections of honeybees undergo numerous reciprocating motions during their lifetime. However, the overlapped contact areas adjacent to the abdominal sections have a shallow wear extent, a physical mechanism that remains obscure to date. Therefore, this study explored a biofrictional reduction model based on a solid surface texture and the hairy surface of the honeybee abdomen. We collected honeybee samples and observed their abdomens using a camera (Zeiss Stemi 508). Subsequently, we sliced these samples using a microtome and detected their microscopic friction. The exterior surface of the honeybee abdomen was not smooth but was distributed with a dense microvilli structure, which played a vital role in adjusting the friction reduction characteristics between the abdominal sections. When the adjacent abdominal sections moved relatively to each other, their upper and lower surfaces were not in direct rigid contact. Briefly, this study shows that the microscale hair arrays on the surface of the posterior abdominal segment can significantly reduce real contact area and friction, which considerably decreases wear or abrasion. The friction reduction mechanism alleviates the abrasion during the relative bending movement and saves a large amount of energy, which is essential for the honeybees' daily activities. This microtexture compliance friction reduction characteristic could be used to fabricate hierarchical surfaces for long-lasting friction reduction mechanisms, which increase the life of soft devices, including soft actuators and hinges.

Paraffin histology is one of the most important and commonly used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.Die paraffinbasierte histologische Untersuchung ist eines der wichtigsten angewendeten Laborverfahren in der diagnostischen Histopathologie. Die Entwicklung der Paraffineinbettung wird oft dem Pathologen Edwin Klebs zugeschrieben. Klebs orientierte sich an den Arbeiten von Stricker, der Embryonen in eine Mischung aus heißem Tristearin und weißem Bienenwachs einbettete, und experimentierte mit Paraffin, um Tumorgewebe einzubetten. Er verwarf den Ansatz aber rasch, da Paraffin das Gewebe nicht infiltrierte. Klebs stand im Briefwechsel mit dem Embryologen Wilhelm His Senior, der von dessen Experimenten erfuhr und beschloss, sich an der Paraffineinbettung zu versuchen. His entwässerte Hühnerembryonen in Alkohol, klärte sie mit Lavendelöl und träufelte heißes Paraffin darüber. Dieses Verfahren ermöglichte ihm die Anfertigung guter Schnitte. In der vorliegenden Arbeit haben wir His’ Protokoll der Paraffineinbettung nachvollzogen, um zu ermitteln, ob His tatsächlich die Infiltrationseinbettung mit Paraffin gelungen ist. Wir befolgten das Protokoll aus seiner 1868 erschienenen Monografie. Anhand der Anweisungen von His gelang es uns nicht, Schnitte mit der von ihm illustrierten Qualität herzustellen. In der Regel zerfiel das Gewebe beim Schneiden wegen schlechter Wachsinfiltration. Brauchbare Schnitte wurden nur bei Abwandlung von His’ Protokoll durch das Wiedereinschmelzen der eingebetteten Embryos in frischem Paraffin erreicht. Möglicherweise ist es uns nicht gelungen ist, das Protokoll von His detailgetreu zu replizieren, oder das Protokoll war unvollständig. Nach unserer Einschätzung hat His wahrscheinlich die Infiltrationseinbettung mit Paraffin erfunden und perfektioniert, aber kein vollständiges Protokoll veröffentlicht.

The macro- and microscopic structure of the lungs of mature rabbits – Oryctolagus cuniculus L., 1758 – European rabbit was studied. This work was performed using comprehensive research methods: anatomical, organometric, histological and histometric. The research was carried out in the conditions of the laboratory of pathomorphology in the Department of Anatomy and Histology. The scientific work was performed on the subject of research work of the Department of Anatomy and Histology of Polissya National University on the theme: “Development, morphology and histochemistry of animal organs in normal and pathological”, according to the state registration number – № 0113V000900. The morphological studies followed the basic rules of Good Laboratory Practice GLP (1981), the provisions of the “General Ethical Principles of Animal Experiments”, adopted by the First National Congress of Bioethics (Kyiv, 2001) and the requirements for the “Rules for carrying out work with experimental animals”, approved by the order of the Ministry of Health № 281 of November 1, 2000 “On measures to further improve the organizational forms of work with the use of experimental animals”. The material for histomorphological studies were pieces of lungs selected from clinically healthy adult rabbits (n = 5). Slices were made from paraffin blocks on a sled microtome MS–2. The thickness of histological sections did not exceed 8–12 μm, which were then stained with hematoxylin and eosin and Van Gizon to study the morphofunctional activity of cells and tissues of the respiratory tract. Histometric studies of histological specimens were performed using a microscope “Micros” with a digital camera at a magnification of × 100–1000. Studies have shown that macroscopically the lungs of mature rabbits have a reflection of the shape of the thoracic cavity, and gradually expands ventrally. The right and left lungs are surrounded by pleural right and left sacs. The pleural cavities of both lungs in the test animals are not interconnected. Organometry showed that the absolute lung mass of rabbits is 18.05 ± 1.32 g, relative to 0.624 ± 0.013 %. The histoarchitectonics of the lungs is formed by the lung lobes. The respiratory department of the lungs is represented by a set of acinuses. The latter are represented by respiratory bronchioles, alveolar passages, alveolar sacs and alveoli that form the alveolar tree. The results of morphometric studies showed that the respiratory (respiratory) part of the lungs of rabbits occupies 39.6 ± 0.62 %, and the connective tissue base, respectively, 58.5 ± 1.27 %. The average volume of alveoli, which are divided into small, medium and large, is 42.3 ± 4.35 thousand μm3. Thus, studies of the macro- and microscopic structure of the lungs of experimental animals are of great general biological importance, as they are an important criterion for objective assessment for the differential diagnosis of diseases of various origins.

Objective: The aim of this study was to assess the micro-push-out bond strength of a mineral-based root canal sealer, BioRoot RCS in canals prepared by K3XF rotary systems of two different tapers. Material and Methods: Eighty caries free maxillary central incisors were used in this study. The samples were allocated into 4 groups (n=20) according to the root canal sealer and taper of the rotary instruments. The samples were obturated using single cone obturation technique. From each root 1mm thick slices at coronal, middle and apical thirds were collected using hard tissue microtome under continuous water coolant. Push-out tests were done for these sections using a Universal testing machine (INSTRON 8801) at a crosshead speed of 1mm/min. One-way analysis of variance (ANOVA) was used to compare the bond strengths within groups and Tukey’s multiple post hoc analysis was used for pair-wise comparison of bond strengths. Results: AH Plus exhibited higher micro-push-out bond strength than BioRootRCS though they did not differ significantly (p>0.05). Preparation of root canals with 6% taper rotary instruments showed higher bond strength than 4% though they did not differ significantly (p>0.05). Conclusion: There was no significant difference between micro-push-out bond strength values of BioRoot RCS and AH Plus. The bond strength values were high in 6% taper canals than 4% canals though the difference was not significant statistically.

Evaluation of the biomechanical properties of soft tissues by measuring the stress-strain relationships has been the focus of numerous investigations. The accuracy of stress depends, in part, upon the determination of the cross-sectional area (CSA). However, the complex geometry and pliability of soft tissues, especially ligaments and tendons, make it difficult to obtain accurate CSA, and the development of CSA measurement methods of soft tissues continues. Early attempts to determine the CSA of soft tissues include gravimetric method, geometric approximation technique, area micrometer method, and microtomy technique. Since 1990, a series of new methods have emerged, including medical imaging techniques (e.g. magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound imaging (USI)), laser techniques (e.g. the laser micrometer method, the linear laser scanner (LLS) technique, and the laser reflection system (LRS) method), molding techniques, and three-dimensional (3D) scanning techniques.

Objective: The teeth play important roles in food mastication, prehension and defense against predators. Although several studies demonstrated the development of teeth in different mammalian species, no data are, to our knowledge, available in Egyptian buffalos. Therefore, the present investigation was conducted to study the development of incisor teeth in buffaloe. Design: Descriptive study. Animals: seventeen buffalo embryos and fetuses of both sexes were used. Their crown vertebral rump lengths (CVRL) ranged from 2 to 46 cm (equivalent to 38 - 177 days old). Procedure: Embryos and fetuses were fixed in 10% neutral buffered formalin and decalcified by 14% EDTA solution for several weeks. The samples were dehydrated, cleared and embedded in paraffin wax using standard techniques. Sections were cut on Leitz microtome and mounted on uncoated slides. For general histological structure, a selection of slides was routinely stained with haematoxylin–eosin and examined by the light microscope. Result: The present study reported for the first time that the prenatal development of incisors in buffalo passes through three sequential stages: the bud, cap and bell stages. Although each stage was easily differentiated based on its form and its distinct developmental features, the three stages were overlapped with each other’s. Generally, the bud stage was observed in 11 cm CVRL buffalo fetuses, meanwhile the cap and bell stages were detected in 23 cm and 30 cm CVRL fetuses respectively. Conclusion and clinical relevance: These results can be used as inductive index for determination of the ages of buffalo embryos and fetuses.

Objective The aim of this study was to compare the microstructure of blood vessels and nerve fibers between different acupoints in the rat skin from the perspective of histology. Methods The skin tissue at “Qūze” (曲泽 PC3)” “Daling (大陵 PC7)” and “Zhōngchōng (中冲 PC9)” regions was taken from the rat forelimb along the pericardium meridian, and cut sagittally with a freezing microtome. After that, the skin sections were stained by fluorescent histochemistry and immunohistochemistry with phalloidin and calcitonin gene-related peptide (CGRP) to reveal the microstructure of blood vessels and nerve fibers respectively, and then examined under a laser scanning confocal microscope. Results The microstructure of blood vessels and nerve fibers was clearly labeled with phalloidin and CGRP, respectively. The blood vessels and nerve fibers were observed in the dermis of “PC3” “PC7” and “PC9” regions, where the bundle of nerve fibers distribute in parallel to the blood vessels, and sent out thin branches to surround the wall of the blood vessels. In contrast, the density of blood vessels and nerve fibers in “PC3” “PC7” and “PC9” were presented orderly in an increased tendency. Conclusion The results of present study demonstrate the differences of the neurovascular microstructure among “PC3” “PC7” and “PC9” in the rat, providing a histological view to insight into the specificity of different acupoints.

Decalcification of mineralized samples for microscopic analysis involves competing factors including decalcification time, preservation of tissue integrity and cost. We investigated the utility of different decalcification solutions for studying joints in AG/WT, BALB/c, C57, DBA1/J mice and Wistar rats. The hind paws of the rodents were removed and fixed with 10% buffered formalin. Specimens were divided randomly into three groups for demineralization: 10% nitric acid, 12.5% EDTA at room temperature and 12.5% EDTA at 35 °C with shaking. Sections of joints were stained with hematoxylin and eosin (H & E). We evaluated decalcification time and expense, ease of cutting sections, preservation of nuclear basophilia and intranuclear detail, and intensity of eosin staining. The 10% nitric acid solution produced the most rapid decalcification for the mice, but not the rats. The 12.5% EDTA solution at 35 °C with shaking did not decrease decalcification time. Effects on microtomy were variable as were the effects on H & E staining. The EDTA solution provided the best basophilia and intranuclear detail for the mice. For rats, only 12.5% EDTA at 35 °C with shaking produced good preservation. Preservation of nuclear basophilia and intranuclear detail for rats was best with 10% nitric acid and EDTA 35 °C. For mice, 10% nitric acid failed to preserve nuclear basophilia and intranuclear detail. For intensity of eosin staining, EDTA at room temperature and EDTA 35 °C was best for both mice and rats. Sections also exhibited good H & E staining in most samples decalcified with 10% nitric acid. Although we found considerable variation among groups of animals, we found less variation among the different mouse strains than between mice and Wistar rats.

Mast cells, known as pro-inflammatory effector cells, are immunocytes present in the meninges and may be involved in the pathophysiology of migraine. This study aims to evaluate the histomorphometric parameters of mast cells located in the convexity of the human intracranial dura mater. For this, samples of intracranial dura mater from eight human fresh cadavers were collected between 8- and 24-h post-mortem. The whole samples were fixed and, subsequently, two fragments of 1.5 cm² each were cut from four different areas of the dura mater convexity, containing a segment of the middle meningeal artery, totaling 64 fragments. After histological processing, the fragments were submitted to microtomy (5 and 10 µm), stained with toluidine blue (0.1%), or immunohistochemically labeled for tryptase, and analyzed using optical microscopy. The following histomorphometric parameters were evaluated: distance from mast cells to vessels, the density of mast cells, and percentage of mast cells with degranulation. Histomorphometric analyzes showed a higher density of mast cells in the vicinity of blood vessels (arterial and venous), with distances around 0-150 µm. A greater number of mast cells was detected near venous vessels in the periosteal layer (17.0 ± 10.1 cells/mm²) than in the meningeal layer (14.1 ± 7.0 cells/mm²) (p < 0.05). Mast cells from the region close to the superior sagittal sinus were found in greater quantity close to the venous vessels (16.7 ± 10.1 cells/mm²) than to the arterial vessels (11.2 ± 7.5 cells/mm²) (p < 0.05). In short, in the convexity of the human intracranial dura mater, mast cells are located close to blood vessels, with a greater number of cells next to the venous vessels of the periosteal layer and in the proximal region of the superior sagittal sinus.

The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.

This study aimed to examine the penetration depth into dentinal tubules of some chelating agents. The 17% EDTA and two preparations containing surfactants (Smear Clear, Bioakt Endo) were tested. Surface tension and liquid viscosity were measured using a Dynamic Contact Angle Analyzer and a Haake rotational rheometer. To measure the penetration depth inside dentinal tubules, thirty maxillary central incisors were selected from a pool of extracted human permanent teeth and allocated to three experimental groups (10 samples each), as well as were mechanically shaped and cleansed with 5.25% NaOCl, followed by each of the chelators being labeled with 0.1 wt % Rhodamine B according to final irrigation protocol established. The samples were embedded in an epoxy resin, after which 200 μm thick transverse sections were obtained at 2, 5, and 8 mm from the apex with a saw microtome. The specimens were then observed using a confocal laser microscope (CLSM) and the penetration of the labeled solution was measured in every third of each sample. Statistical analysis was performed using ANOVA or Kruskal–Wallis tests according to the distribution of data, evaluated with the Shapiro–Wilk normality test. Viscosity and surface tension tests have shown that BioAKT Endo has the lowest values compared to EDTA and Smear Clear. The medium penetration depth did not significantly differ among the three irrigants, while it increased considerably from the apical to the coronal level in all groups. Additionally, the maximum penetration depth increased significantly from the apical to coronal level, while among groups, BioAKT Endo showed the highest values at the apical and middle level compared to the other irrigants. No significant differences were observed among the three groups in medium and maximum penetration depths when the entire root was considered. New irrigants containing surfactants show reduced surface tension and, in one case (BioAKT Endo), viscosity. The lowering of the surface tension allows for better penetration of liquids into dentinal tubules than EDTA alone, thus improving the cleaning of the root canal system.

The inferior lobes are prominent bilateral brain areas in the hypothalamus of neopterygians among the ray-finned fishes. They are known as multisensory integration centers. As such, they should play a major role in fish evolution. In this study, a comparative morphometric analysis was performed. The morphology of the hypothalamus, where the inferior lobe is considered as fully developed first in Lepisosteus, was then re-examined. One hundred brains from different species of 60 families of ray-finned fishes were stained with cresyl violet and embedded in methacrylate. They were then cut on a microtome while conducting block-face imaging. The volumes were determined for the whole brain, brain areas, and nuclei. Since visual input represents a major sensory input for the inferior lobe, the nucleus glomerulosus, a visual-related nucleus in paracanthopterygian and acanthopterygian teleosts, and the tectum opticum were included in the investigations. The morphometric analysis revealed that the relative volume of the inferior lobes increases significantly from species of the Lepisosteiformes to the Tetraodontiformes. In addition, a positive correlation was detected between the relative volume of the inferior lobes and either the relative volume of the nucleus glomerulosus or the relative volume of the tectum opticum. These correlations, in combination with findings from previous hodological and behavioral studies, give rise to the speculation that the inferior lobes may be involved in higher cognitive processes and complex social interactions.

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell-fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as "nucleolus-like body" (NLB) was detected inside the germinal vesicle in PO3- and CN- ions generated by nucleic acids and proteins respectively. Significant difference in PO3- intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3- and CN- ion images is 12.9 ± 0.2 μm and 11.9 ± 0.2 μm respectively, which suggests that bright halo of PO3- ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0-2.5 μm size inside nucleoplasm with increased PO3- and CN- signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes.

Death of osteocytes is synonymous of bone death. Aseptic osteonecrosis of the femoral head is a lesion characterized by the death of osteocytes occurring after major vascular changes. The evolution may lead to hip osteoarthritis, which requires total hip arthroplasty in most cases. Evolution of aseptic osteonecrosis in four radiological stages is well known. We analyzed 24 femoral heads from patients with osteonecrosis or osteoarthritis, retrieved at the time of surgery for a hip arthroplasty. The aim of the study was to clearly identify the necrotic bone from the living bone in the histological samples. The femoral heads were sawed, and a large sample was harvested in the superior zone; it was stained en-bloc with rhodamine dissolved in formalin to make the osteocytes fluorescent under UV light microscopy. Undecalcified sections, 7 μm thick, were obtained on a heavy-duty microtome. A micrographic analysis using two UV excitation wavelengths visualized the living osteocytes (in green) and the bone matrix (in blue). A simple method to prepare combined images is described. In addition, the blocks can be analyzed by confocal microscopy to visualize more details. It is possible to identify at low magnification the osteocytes within the bone matrix and the osteonecrotic areas where osteocytes have disappeared. Identification of osteocytes showed that newly formed bone packets are laid on dead trabeculae in patients with aseptic osteonecrosis or with osteoarthritis. In the osteosclerotic areas, the enlarged trabeculae have a dead central core surrounded by recently apposed bone structure units.

Obtaining high-quality sections of the nail plate poses a significant challenge to histopathology technicians world over. Nail is a heavily keratotic hard tissue that tends to split or tear while sectioning when processed and embedded in a routine manner. Many agents such as phenol, alcohol, and thioglycolate have been tried for the purpose of softening a variety of experimental materials. However, there is no clear consensus on any single agent. The study was conducted with the aim of evaluating and comparing the role of various compounds as softening agents for nail biopsies with inflammatory disease. Thirty paraffin-embedded nail biopsies were subjected to four softening agents: distilled water (DIH20), 30% potassium hydroxide (KOH), hair removal cream, and fabric conditioner. The ease of sectioning, the incidence of juddering (i.e. 'venetian blind' effect), and the shattering of tissue were recorded. Hematoxylin and eosin-stained sections were examined microscopically. Sectioning was very easy after using fabric conditioner, with good quality sections, and hair removal cream produced comparable results. The incidence of juddered, shattered sections after using hair removal cream was considerably higher (63.33%) compared to fabric conditioner-treated nails (16.67%). Microtomy of nail biopsies was found to be easiest after using 30% KOH with moderate section quality. DIH2O could neither allow easier sectioning nor obtain good sections for interpretation. Fabric conditioner and hair removal cream proved to be the effective keratin softeners, while 30% KOH worked effectively when the nail plate alone was submitted for histological examination.

Rapid histologic assessment of fresh prostate biopsies may reduce patient anxiety, aid in biopsy sampling, and enable specimen triaging for molecular/genomic analyses and research that could benefit from fresh tissue analysis. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can produce high-resolution images of freshly excised tissue resembling formalin-fixed paraffin-embedded (FFPE) H&E. NLM enables evaluation of tissue up to ~100 µm below the surface, analogous to serial sectioning, but without requiring microtome sectioning. One hundred and seventy biopsies were collected from 63 patients who underwent in-bore MRI or MRI/ultrasound fusion biopsy procedures. Biopsies were stained in acridine orange and sulforhodamine 101, a nuclear and cytoplasmic/stromal fluorescent dye, for 45 s. Genitourinary pathologists evaluated the biopsies using NLM by translating the biopsies in real time to areas of interest and NLM images were recorded. After NLM evaluation, the biopsies were processed for standard FFPE H&E and similarities and differences between NLM and FFPE H&E were investigated. Accuracies of NLM diagnoses and Gleason scores were calculated using FFPE histology as the gold standard. Pathologists achieved a 92.4% sensitivity (85.0–96.9%, 95% confidence intervals) and 100.0% specificity (94.3–100.0%) for detecting carcinoma compared to FFPE histology. The agreement between the Grade Group determined by NLM versus FFPE histology had an unweighted Cohen’s Kappa of 0.588. The average NLM evaluation time was 2.10 min per biopsy (3.08 min for the first 20 patients, decreasing to 1.54 min in subsequent patients). Further studies with larger patient populations, larger number of pathologists, and multiple institutions are warranted. NLM is a promising method for future rapid evaluation of prostate needle core biopsies.

Cytology specimens and biopsy tissues are frequently small and pale, making them difficult to visualize grossly in paraffin. Ten dyes were assayed on small tissues to determine if specimen discernibility could be increased during the embedding and microtomy steps in the histological process. The ideal dye should not remain visible in a tissue section microscopically after subsequent staining and must not interfere with immunohistochemistry (IHC) assays. This study found that Harris hematoxylin and 1% aq. toluidine blue solution were the best labelers for gross tissue visualization and did not adversely affect post-processing staining and IHC assays.

Despite tremendous advancements in in vivo imaging modalities, there remains substantial uncertainty with respect to tumor delineation on in these images. Histopathology remains the gold standard for determining the extent of malignancy, with in vivo imaging to histopathologic correlation enabling spatial comparisons. In this review, the steps necessary for successful imaging to histopathologic correlation are described, including in vivo imaging, resection, fixation, specimen sectioning (sectioning technique, securing technique, orientation matching, slice matching), microtome sectioning and staining, correlation (including image registration) and performance evaluation. The techniques used for each of these steps are also discussed. Hundreds of publications from the past 20 years were surveyed, and 62 selected for detailed analysis. For these 62 publications, each stage of the correlative pathology process (and the sub-steps of specimen sectioning) are listed. A statistical analysis was conducted based on 19 studies that reported target registration error as their performance metric. While some methods promise greater accuracy, they may be expensive. Due to the complexity of the processes involved, correlative pathology studies generally include a small number of subjects, which hinders advanced developments in this field.

Cell-staining studies on mammalian tissue are often performed on frozen sections. This is the gentlest method for the preparation of samples and gives good preservation of cell structure and antigens. Its principal disadvantages are that the specimens must be stored frozen and a special microtome, known as a cryostat, is required. In addition, many clinical specimens are not available in this form, and most classical histological descriptions of tissue structure and pathology are based on the use of formalin-fixed, paraffin-embedded (FFPE) material.

We describe 3-dimensional histology of fresh or fixed unimbedded tissue using a vibrating microtome with MUSE (microscopy with UV surface excitation) to acquire block-face images that can be reconstructed into 3-dimensional data sets.

Histological processing of mineralised tissue (e.g. bone) allows examining the anatomy of cells and tissues as well as the material properties of the tissue. However, resin-embedding offers limited control over the specimen position for cutting. Moreover, specific anatomical planes (coronal, sagittal) or defined landmarks are often missed with standard microtome sectioning. Here we describe a method to precisely locate a specific anatomical 2D plane or any anatomical feature of interest (e.g. bone lesions, newly formed bone, etc.) using 3D micro computed tomography (microCT), and to expose it using controlled-angle microtome cutting. The resulting sections and corresponding specimen's block surface offer correlative information of the same anatomical location, which can then be analysed using multiscale imaging. Moreover, this method can be combined with immunohistochemistry (IHC) to further identify any component of the bone microenvironment (cells, extracellular matrix, proteins, etc.) and guide subsequent in-depth analysis. Overall, this method allows to:•Cut your specimens in a consistent position and precise manner using microCT-based controlled-angle microtome sectioning.•Locate and expose a specific anatomical plane (coronal, sagittal plane) or any other anatomical landmarks of interest based on microCT.•Identify any cell or tissue markers based on IHC to guide further in-depth examination of those regions of interest., Graphical abstract Multiscale imaging of the desired anatomical location and plane based on correlative microscopy and histology.Image, graphical abstract

The capillary network of the kidney glomerulus filters small molecules from the blood. The glomerular 3D structure should help to understand its function, but it is poorly characterized. We therefore devised a new approach in which an automated tape collecting microtome(ATUM) was used to collect 0.5 micron thick serial sections from fixed mouse kidneys. The sections were imaged by scanning electron microscopy at ∼50 nm / pixel resolution. With this approach, 12 glomeruli were reconstructed at an x-y-z resolution ∼10x higher than that of paraffin sections. We found a no-cross zone between afferent and efferent branches on the vascular pole side; connections here could allow blood to exit without being adequately filtered. Network analysis indicates that the glomerular network does not form by repetitive longitudinal splitting of capillaries. It also suggests that capillaries vary their diameter to make flow more efficient. The shortest path (minimum number of branches to travel from afferent to efferent arterioles) is relatively independent of glomerular size and is present primarily on the vascular pole size. This suggests that the shortest path is established on the vascular pole side, after which new branches and longer paths form on the urinary pole side. Thus the 3D structure of the glomerular capillary network provides useful information with which to understand glomerular function. Other tissue structures in the body may benefit from this new three dimensional approach.

ObjectiveThis study aimed to investigate the direct monosynaptic projections from cortical functional regions to the cerebrospinal fluid (CSF)-contacting nucleus for understanding the functions of the CSF-contacting nucleus.MethodsThe Sprague–Dawley rats received cholera toxin B subunit (CB) injections into the CSF-contacting nucleus. After 7–10 days of survival time, the rats were perfused, and the whole brain and spinal cord were sliced under a freezing microtome at 40 μm. All sections were treated with the CB immunofluorescence reaction. The retrogradely labeled neurons in different cortical areas were revealed under a confocal microscope. The distribution features were further illustrated under 3D reconstruction.ResultsThe retrogradely labeled neurons were identified in the olfactory, orbital, cingulate, insula, retrosplenial, somatosensory, motor, visual, auditory, association, rhinal, and parietal cortical areas. A total of 12 functional areas and 34 functional subregions showed projections to the CSF-contacting nucleus in different cell intensities.ConclusionAccording to the connectivity patterns, we conclude that the CSF-contacting nucleus participates in cognition, emotion, pain, visceral activity, etc. The present study firstly reveals the cerebral cortex→CSF-contacting nucleus connections, which implies the multiple functions of this special nucleus in neural and body fluid regulations.

This protocol describes how visualize internal structures, from mandibular mycangia, to beetle heads or bodies. This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Aim: Evaluation of the impact of three different obturation techniques on the penetration depth and adaptation of a bioceramic root canal sealer(BioRoot RCS) to root dentin using scanning electron microscope and confocal laser scanning microscope. Materials and Methods: Recently extracted ninety mandibular premolar teeth of similar dimensions (±0.2 mm) with single canals and straight roots were segregated into three experimental groups (n = 30 each) based on the method of obturation used, i.e., lateral compaction technique, warm vertical compaction technique, and injectable thermoplasticized technique. After obturation, half of the root samples (n = 15) in each group were sectioned horizontally by hard tissue microtome at 3, 6, and 9 mm, respectively, from root apex for measuring sealer penetration depth with a confocal laser scanning microscope. Longitudinal sections of the samples (n = 15) were used for measuring the adaptation of sealers to radicular dentin using a scanning electron microscope. Statistical Analysis: Intergroup comparison of sealer penetration depth and adaptation to root dentin was analyzed using one-way ANOVA, and pairwise comparisons were made using Tukey's post hoc test. Results: Warm vertical condensation technique showed deeper root dentin penetration of the sealer. Better adaptation of the sealer to apical root dentin with minimal voids (P ≤ 0.05) was achieved with warm vertical condensation in comparison to the injectable gutta-percha method and lateral condensation obturating techniques. Conclusion: At all three root levels (coronal, middle, and apical), warm vertical condensation technique exhibited more sealer penetration with minimum gaps, whereas lateral condensation technique exhibited more gaps and less sealer penetration.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

Objective To investigate whether the CSF-contacting nucleus receives brainstem and spinal cord projections and to understand the functional significance of these connections. Methods The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in Sprague-Dawley rats according the previously reported stereotaxic coordinates. After 7-10 days, these rats were perfused and their brainstem and spinal cord were sliced (thickness, 40 μm) using a freezing microtome. All the sections were subjected to CB immunofluorescence staining. The distribution of CB-positive neuron in different brainstem and spinal cord areas was observed under fluorescence microscope. Results The retrograde labeled CB-positive neurons were found in the midbrain, pons, medulla oblongata, and spinal cord. Four functional areas including one hundred and twelve sub-regions have projections to the CSF-contacting nucleus. However, the density of CB-positive neuron distribution ranged from sparse to dense. Conclusion Based on the connectivity patterns of the CSF-contacting nucleus receives anatomical inputs from the brainstem and spinal cord, we preliminarily conclude and summarize that the CSF-contacting nucleus participates in pain, visceral activity, sleep and arousal, emotion, and drug addiction. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the brainstem and spinal cord, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulation.

Atrioventricular node is responsible for delaying the passage of the electrical impulse to ventricles in order to protect them from fast depolarizations coming from the atria. The importance of this study is to identify the morphological variations of the components of atrioventricular zone that affect the conduction system and its clinical relationship in different species of mammals. We analyzed ten human hearts, nine from horses, eight from pigs, and five from dogs without a clinical history of cardiac pathologies. Histological section thickness of 5 μm were obtained with a microtome and stained with hematoxylin–eosin and Masson's trichrome. We observed both an increase in collagen fibers and a decrease in the size of P cells (nodal pacemaker cells) within the atrioventricular node in dogs, horses and pigs in cases that presented cartilage in fibrous body. The percentage of fundamental substance in atrioventricular node was significantly higher in dogs and the percentage of collagen fibers was higher in pigs, both than in humans. The presence of cartilaginous metaplasia in cardiac fibrous skeleton from different species decreases the size of atrioventricular node and its cells and increases the percentage of collagen fibers within the node, which can reduce the transmission of the electrical impulse to ventricles and therefore predispose to the presentation of ventricular arrhythmias. Morphometric analysis has allowed us to objectively quantify each of the components of AV node and compare them in the different species.

Objective: To investigate the projections the cerebrospinal fluid-contacting (CSF-contacting) nucleus receives from the diencephalon and to speculate on the functional significance of these connections. Methods: The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in SD rats according to the experimental formula of the stereotaxic coordinates. Animals were perfused 7–10 days after the injection, and the diencephalon was sliced at 40 μm with a freezing microtome. CB-immunofluorescence was performed on all diencephalic sections. The features of CB-positive neuron distribution in the diencephalon were observed with a fluorescence microscope. Results: The retrograde labeled CB-positive neurons were found in the epithalamus, subthalamus, and hypothalamus. Three functional diencephalic areas including 43 sub-regions revealed projections to the CSF-contacting nucleus. The CB-positive neurons were distributed in different density ranges: sparse, moderate, and dense. Conclusion: Based on the connectivity patterns of the CSF-contacting nucleus that receives anatomical inputs from the diencephalon, we preliminarily assume that the CSF-contacting nucleus participates in homeostasis regulation, visceral activity, stress, emotion, pain and addiction, and sleeping and arousal. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the diencephalon, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulations.

The structural and functional features of the tongue attract the attention of researchers who are often interested in the peculiarities of the structural organization of its arterial bed, which plays an important role in blood supply and morphogenesis of the organ in various injuries. The aim of the research was to study the age-specific features of remodeling of tongue arteries in the experimental animals. The materials of the study were the arterial vessels of the tongue of 30 laboratory albino male rats, which were under normal vivarium conditions. The test animals were divided into two groups. The 1st group consisted of 15 intact, practically healthy animals at the age of 8 months, the 2nd -15 intact rats at the age of 24 months. The euthanasia of experimental animals was carried out by bloodletting under conditions of thiopental-sodium anesthesia. The pieces were cut from the middle part of the tongue and fixed in a 10% neutral solution of formalin. The indicated pieces of tongue were carried out through the ethyl alcohol of increasing concentration and placed in paraffin blocks. After deparaffinization microtome sections 5-7 microns thick were stained with hematoxylin-eosin, by van Gizon, Mallory, Weigert, and toluidine blue. The morphometry of the arteries of the middle (external diameter 51-125 microns) and small calibers (outer diameter – 26-50 microns) of the tongue was carried out. The morphometry of the arteries of the tongue was determined by their external and internal diameters, the thickness of the mediums and adventitia, the height of the endothelial cells, their nuclei, the nuclear-cytoplasmic relations in the endothelial cells, the Kernogan index, and the relative volume of the damaged endothelial cells. Quantitative indicators were processed statistically. The analysis of the obtained morphometric parameters revealed that with the age the arteries of the small caliber have changed more. The outer diameter of arteries of small caliber tongue of the 24-month-old albino rats has increased only for 1.2% and the clearance has decreased by 4.4%. An increase in the outer diameter of the examined vessels and a decrease in their lumen led to a change in the Kernogan index, which with a statistically significant difference (p

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.

The aim of this study was to evaluate the ability of spectral-domain optical coherence tomography (SD-OCT) to display the roof of the pulp chamber and to estimate the residual dentin thickness (RDT) of the pulp complex. The roots of 20 extracted human molars were embedded in epoxy resin, and crowns were longitudinally sectioned in the mesial-distal direction, exposing the pulp chamber. The coronal part of the crown was removed up to an RDT to the pulp chamber roof of 2 mm. Samples were imaged by SD-OCT from coronal view and by light microscopy (LM) in the sagittal plane. Using a microtome, dentin was subsequently removed in four levels from the occlusal aspect in steps of 250 μm. At each level, RDT was documented and measured by both methods. The data were compared (Spearman’s rho correlation coefficient, Wilcoxon signed-rank test). Using OCT, the roof of the pulp chamber was first displayed at a maximum RDT of 1.94 mm. The minimal RDT that could be imaged by OCT was 0.06 mm. Values from both methods were strongly correlated (r, 0.83–0.95; pi ≤ 0.05) and differed significantly for large RDTs (dentin levels 1, 2; pi

The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin histologic evaluations. There are few studies that have reported detailed processes for preparing 3-D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining and light microscopic evaluations.

This study aimed to determine the effects ofglyphosate, a herbicide commonly used in weed control,on aquatic life. For this purpose, 30 one-year-oldrainbow trout with an average weight of 150-165 g wereobtained from a local trout production station inMazmanlı (Hatay, Turkey) and transferred to ourlaboratory, where they were allowed to adapt to the newenvironment in polyethylene tanks approximately for 7days.Three groups each containing 10 fish were formed: acontrol group with no treatment, the group treated with1.25 mg/l glyphosate-based herbicide (GBH), and thegroup treated with 2.5 mg/l glyphosate-based herbicide.At the end of 14 days of treatment, blood samples weretaken from the caudal vein of the fish under anaesthesia,and their sera were separated. Total oxidant/antioxidantlevels and paraoxonase activity were analysed in theobtained serum samples. Also, for histopathologicalexamination, gill tissues were removed and fixed in10% buffered formalin. After the fixation and routinetissue processing (graded alcohols, methyl benzoate andbenzol processing), the tissues taken were embedded inparaffin and 5 μm serial sections were taken bymicrotome from the blocks to slides pre-coated withchrome alum gelatine. Histopathological changes wereexamined at the light microscopic level by staining thesections with haematoxylin-eosin. According to theevaluation of the biochemical parameters obtained fromthe groups, antioxidant capacity and paraoxonaseactivity decreased and oxidant level increased in thegroup treated with 1.25 and 2.5 mg/l glyphosate-basedherbicide as compared with the control group. In thehistopathological examination of the sections, it wasobserved that the primary and secondary lamellae had anormal structure in the gill sections obtained from thecontrol group. Irregular secondary lamellae andepithelial hyperplasia were observed in the gill sectionsobtained from the group receiving 1.25 mg/l GBH. Andin the gill sections obtained from the group receiving2.5 mg/ l GBH, swelling in chloride cells, degenerationin secondary lamellae, and areas of necrosis weredetected. In conclusion, results of the study suggeststhat glyphosate, which is widely used in agriculturalactivities and has a potential to leak into aquaticecosystems, may cause oxidative stress due to reactiveoxygen species formed in the rainbow trout, and maytherefore has a toxic effect.

Embedding of tissue samples that maintains a desired orientation is critical for preparing sections suitable for diagnosis and study objectives. Methods to prepare tissue sections include: (i) paraffin embedding or snap-freezing followed by microtome or cryostat sectioning; and (ii) agarose embedding followed by cutting on a vibrating microslicer. Although these methods are useful for routine laboratory work, preparation of small and fragile tissues such as mouse organs, small human biopsy samples, and cultured floating spheres is difficult and requires special skills. In particular, tissue specimen orientation can be lost during embedding in molds and subsequent sectioning. Here, we developed a method using low melting temperature (LM) gelatin either alone or mixed with agarose to preliminarily embed collected tissues that are either prefixed or unfixed, followed by conventional fixation, paraffin embedding, freezing, and sectioning. The advantage of the method is that the LM gelatin and its mixture with agarose can be handled at room temperature but quickly hardens at 4°C, which allows embedding, trimming, and arranging of small and fragile tissues in a desired orientation and are compatible with traditional stainings. Thus, this method can have various laboratory applications and can be modified according to the needs of each laboratory.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (similar to 7 days) to elderly (similar to 38 months) rabbits were monitored in intact (n(tendons) = 24) and microtome sectioned (ntendons = 11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Lambda) started at very low values (A = 2.0 +/- 0.6 mu m, Lambda = 19 +/- 4 mu m) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development. The project was mainly supported by the Grant Agency of Charles University (grant no. GAUK 956213), Czech Science Foundation, grant no. 16-14758S, by the Ministry of Health of the Czech Republic, grant no. 16-28637A, by the Ministry of Education, Youth and Sports within National Sustainability Programme I, project no. LO1309, by MEYS (LM2015062 Czech-BioImaging), by ERDF OPPK Brain-View CZ.2.16/3.1.00/21544, and D.H. acknowledges the support from the Flemish Government and Hasselt University for a research stay at Hasselt University.

Background There is limited data on nail histopathology techniques. The objective of this study was to examine nail histopathology techniques currently in use internationally. Methods An online survey was sent to the European Nail Society and Council for Nail Disorders during 2015–16. Results There were fifty seven respondents, from twenty countries comprising dermatologists, podiatrists and pathologists. Specimens were unmarked or marked using ink or a suture and fixed in 10% formalin, from 6 to 48 hours before embedding in paraffin wax (90% (17/19), liquid nitrogen (frozen section, 1/19) and 2-hydroxyethylmethacrylate (plastic, 1/19). Nail softening was undertaken by 71% (17/24) of respondents for 6 to 48 hours using Mollifex Gurr (12.5%, 3/24), 10% potassium hydroxide solution (12.5%, 3/24) or 10% potassium thioglycolate cream (12.5%, 3/24). Sections thickness was 4-9 µm (62.5%), using a steel microtome (92%,12/13) on glass slides (91.6%, 11/12). Hematoxylin and eosin (H&E) was routine for all biopsies and Periodic acid Schiff (PAS) (100%,15/15) for fungus The favored stain for differentiating melanin and hemoglobin was Fontana-Masson (60%, 6/10). For pigmented lesions, Melan-A was always employed by all respondents (9/9). Conclusion Nail histopathology processing has some small variations from normal skin processing.

The structural organization of the biological material in the nucleoli (Nucleolus-Like Bodies (NLBs)) in mouse GV oocytes has been studied with atomic force microscopy (AFM), scanning electron microscopy (SEM), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Experimental samples are prepared by quick plunge freezing, followed by freeze-drying, embedding in Epon epoxy resin, and sectioning with a microtome. The biological material in the NLBs is found to be organized as a domain structure of densely packed granules with sizes from tenths of a micron to a micron.

Context White spot lesions form within 4 weeks of bracket placement and 96% patients show its prevalence during fixed treatment. Less than optimal esthetic result is achieved after orthodontic treatment due to demineralization of enamel adjacent to fixed orthodontic appliances in patients with inadequate oral hygiene. Aims An invitro evaluation of areas of demineralization, before and after subjecting the teeth to caries producing solution for 30 days for three adhesive resins, Transbond XT†, Fuji-Ortho-LC and Filtek Z 350♦. Settings and design 180 sound premolars were divided into 2 broad groups of 90 each. Control group were exposed to artificial saliva solution for 30 days, whereas, experimental group were exposed to both artificial saliva (for 11 h) and caries producing solution (for 1 h) at the rate of 2 cycles per day for a period of 30 days. These groups were further subdivided into six subgroups of 30 teeth each. After 30 days, for both groups, teeth cusps were ground flat and longitudinal bucco-lingual sections were made with a series 1000 Deluxe Hard tissue microtome, photographed under polarized light microscopy and Motic Image plus software 3.2 was used to measure amount and depth of demineralisation. Depth of demineralization lesion was obtained as an average from three sites within the lesion (gingival, middle and occlusal). Results Analysis of variance (two-way ANOVA) and Tukey's HSD post hoc test indicated significant differences among these groups and materials. Teeth bonded with conventional composite (Transbond XT™† 3M), nanofilled restorative material (Filtek Z 350™♦ 3M) showed more lesion depth when compared with RMGI cement (Fuji-Ortho-LC™ ). RMGIC groups had smaller mean lesion depths when compared with composite resin groups. Materials from experimental group showed increased depth of demineralization (p Conclusions Area of demineralization in experimental group increased significantly when compared with control group. Least amount of demineralization was seen with RMGIC (Fuji-Ortho-LC ) and highest with conventional orthodontic composite (Transbond XT†).

To evaluate the impact of the fiber–tissue distance on histological parameters in a porcine kidney model. Four lasers were tested at 60 W using a 600-µm bare-ended fiber: a continuous wave (cw) thulium fiber laser (TFL), a super pulsed (SP) TFL, a Ho:YAG laser, and a blue diode laser (BDL). All tissue samples were mounted on a motorized XY-translation stage. The fiber–tissue distance was changed within a range from 0to 6 mm. Ten incisions were made with each laser at each distance. Afterwards, the tissue samples were sliced with a microtome for lactate dehydrogenase staining to determine zones of thermal damage. In contact mode, the largest incision depth was found for the cw TFL (1.7 ± 0.1 mm) compared to the SP TFL (1.0 ± 0.1 mm), BDL (0.9 ± 0.1 mm) and HoYAG laser (1.1 ± 0.1 mm), respectively. With regard to the coagulative properties, the SP TFL and the Ho:YAG laser showed comparable coagulation depths with 0.7 ± 0.1 and 0.6 ± 0.1 mm, respectively. At 2 mm fiber–tissue distance, the Ho:YAG laser was the only laser that vaporized tissue (incision depth: 0.2 ± 0.1 mm). The BDL was the only laser that caused coagulation at a distance of 3–5 mm. Our results support the clinical observation that cw TFL must be defocused for best coagulation, while the coagulation depth of the SP TFL remains nearly constant within the range of 0–3 mm. Increasing the distance of the laser fiber to the tissue up to 5 mm did not cause significant differences with regard to coagulation depth using the BDL.

This section describes an immunohistochemistry method to analyze interleukin-1 (IL-1) in oral squamous cell carcinoma. The described protocol has been optimized for IL-1 detection in formalin-fixed, paraffin-embedded oral tissue sections by light microscopy. A few common pitfalls and problems associated with immunohistochemical staining are discussed.

Some studies have proven possible potentiating toxic effect of combined pesticides. The potentiating effect may be related to the impact of a certain substance on enzymatic activity of another one. Due to combined preparations popularity, the importance of evaluating probability of summation and synergetic or antagonizing action of the one-purpose substances becomes particularly important. Aim of the researchisto studypeculiarities of histomorphological liver changes associated with combined effect of active substances with one-purpose action. Methods.The authors have used toxicological, histochemical and statistical study methods. 45 rats have been used for the study. The liver specimens for histochemical analysis were selected immediately after dissection of the rats, from which the 5 micrometer sections were prepared, using freezing microtome. After leaving the sections in cold acetone, the histochemical reaction on determining the γ-glutamyl transpeptidase, a transformed hepatocyte marker, which forms nodules while proliferates, was conducted. Results.The authors have established thatthe above-mentioned characteristics inanimals which were administered the drug (benthiavalicarb-isopropyl+folpet) do not differ from negative control group. General specific area of nodules per сm2and specific quantity of nodules per сm2haven’t reliably changed, the control group exceeding these data by 18 % and 12 %, respectively. Discussion.The study of the benthiavalicarb-isopropyl and folpet mixture effect on animals has not revealed liver tissue proliferation of the carcinogen-transformed cells and appearance of hyperplastic nodes, which express the γ-glutamyl transpeptidase, a histochemical marker of pre-tumor changes. So, the fungicide (benthiavalicarb-isopropyl+folpet) has not induced multiplication or size increase of glutamyl transpeptidase nodules, which evidences about loss of the modifying effect of folpet on the benthiavalicarb-isopropyl carcinogenicity in this preparation formula. The obtained results may be used when deciding on administration of combined fungicides with active substances of one-purpose action.

Swelling velocity in dependence on the anatomical cutting direction of yew [Taxus baccata L.] and boxwood [Buxus sempervirens L.] was determined at temperature of 20 °C and at relative humidity of 10% and 100%. The investigations, conducted on a microtome section level, showed a similar behaviour for specimens of both wood species. It was possible to determine that the swelling velocity for yew and boxwood increases in its anatomical cutting directions. The longitudinal direction showed the lowest value, the tangential direction, by distinction, the highest value. Furthermore, a significant influence of early wood and late wood content on the swelling velocity for yew was detected. Keywords: Yew, Boxwood, Microtome, Swelling velocity, Wood tissue, Cutting directions

Optical Coherence Tomography (OCT) is a promising technology for monitoring of pharmaceutical coating processes. However, the pharmaceutical development and manufacturing require a periodic validation of the sensor's accuracy. For this purpose, we propose polyethylene terephthalate (PET) films as a model system, to periodically validate the measurements during manufacturing. This study proposes a new approach addressing the method validation requirement in the pharmaceutical industry and presents results for complementary methods. The methods investigated include direct measurement of the layer thickness using a micrometer gauge as reference, X-ray micro computed tomography, transmission and reflectance terahertz pulsed imaging, as well as 1D- and 3D-OCT. To quantify the significance of OCT for pharmaceutical coatings, we compared the OCT results for commercial Thrombo ASS and Pantoloc tablets with direct measurements of coating thickness via light microscopy of microtome cuts. The results of both methods correlate very well, indicating high intra- and inter-tablet variations in the coating thickness for the commercial tablets. The light microscopy average measured coating thickness of Thrombo ASS (Pantoloc) was 71.0 µm (83.7 µm), with an inter-coating variability of 8.7 µm (6.5 µm) and an intra-coating variability of 2.3 µm to 9.4 µm (2.1 µm to 6.7 µm).