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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%.

Background. The quantity and quality of the etching pattern produced by acids play a significant role in the wettability and contact angle of the adhesive with the enamel surface in orthodontics. The better the etch pattern, the more the surface energy of the enamel, better the penetration of the adhesive, which ultimately results in better bond strength. The present study aimed to evaluate the contact angle of the bonding agent with the enamel surface etched by five commercially available etchants and check if any difference existed between the five. Materials and Methods. Twenty-five human maxillary and mandibular central incisors and premolars extracted for orthodontic or dental purposes were used in this study as samples. The teeth were allocated into five groups based upon the etchant used to etch the enamel surface. After the samples were etched, a hard tissue microtome was used to create thin slices of the enamel surface. The samples were then exposed to bonding agent Ormco Enlight. The bonding agent was released in the form of droplets onto the enamel slices mounted on a contact angle goniometer. The contact angle values were tabulated, and statistical analysis using the one-way ANOVA test was carried out. Results. The contact angle measurements of the etchant group, DPI, were the lowest, while D-tech has the highest contact angle values. However, statistical analysis revealed no statistically significant difference between the contact angle measurements of the five groups included in the study. Conclusion. No statistically significant difference existed between the five etchant groups included in the study. However, commercially available etchant DPI showed lesser contact angle and thus better wettability in comparison to other groups. Further elemental analysis and surface analysis are required to validate these results.

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.

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.

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.

Modern studies of detailed processes in plant development would not be possible without using a wide range of fluorescent proteins (R. Day and M. Davidson, 2009; D. Chudakov et al., 2010). However, application of fluorescent proteins is restricted due to problems with their visualization in plant tissues. Plants are difficult objects for microscopic studies: even the most advanced methods have significant limitations regarding the depth of light penetration due to the scattering and absorption of light by cell walls. Therefore, to study the distribution of reporter fluorescent proteins in large organs typical for most plants, it is necessary to fix the plant material and prepare thick histological sections with a vibrating-blade microtome. Chemicals traditionally used for fixing, dehydrating and embedding of plant tissues samples lead to changes in the structure of fluorescent proteins and, as a result, often to the loss of their fluorescence. Therefore, it is important to optimize the protocols for fixing plant tissues, preparing sections and studying the distribution of fluorescent proteins by laser scanning confocal microscopy. In this work, we have proposed for the first time an integrated approach to fixation of tissues of transgenic plants and preparation of sections in the course of the study of patterns of cellular response to auxin and expression of transcription factors using laser scanning confocal microscopy. Our aim was to sum up modern approaches to the application of an effective universal technique for visualization of tissue and cellular patterns of the fluorescent reporter proteins distribution on sections of large non-model plants. The first step for using fluorescent proteins in plants is the generation of genetic constructs that carry the promoter of the gene of interest fused to a reporter gene encoding a fluorescent protein. In this case, a transformation protocol has to be available for the selected plant species. We have described the use of the Gateway® cloning technology for the construction of vectors for plant transformation that meet modern experimental requirements. In order to study auxin localization in vivo we developed a series of vectors with genes encoding various fluorescent proteins (eGFP, tdTomato, mRuby3) under the control of the auxin-sensitive DR5 promoter (E. Ilina et al., 2012). We now demonstrate the advantage of nuclear-targeted fluorescent proteins (mNeonGreen-H2B, tdTomato-H2B, mRuby3-H2B), as well as the possibility of their application for additional visualization of cell nuclei in combination with highly specific cell wall staining using SCRI Renaissance2200. An effective method for constructing vectors to study cell-specific expression patterns of developmental regulators is presented using the transcription factor genes GATA24 (A. Kiryushkin et al., 2019) and LBD16 in some Cucurbitaceae species as examples. We also applied an expression cassette, pAtUBQ10::DsRED1 (E. Limpens et al., 2004), carrying a gene encoding red fluorescent protein under the control of a constitutive promoter, to demonstrate the advantages of the use of fluorescent proteins in screening for transgenic vs. wild type roots. A new method of fixation and clearing of plant tissues containing reporter fluorescent proteins and preparation of sections is presented for the example of transgenic roots of Cucurbitaceae. The advantage of using melted agarose compared to embedding media for orienting plant parts during the preparation of samples was demonstrated. The increase of the photostability of fluorescent proteins in sections due to the use of clearing reagent ClearSee (D. Kurihara et al., 2015) as a mounting medium is demonstrated. Altogether, we present a combination of the advantages that are provided by application of a complex of several modern methodical approaches of sample preparation and laser scanning confocal microscopy that offers significant advantages for studying of the developmental biology of large non-model plants.

Synchrotron radiation-based infrared microspectroscopy (SR-IMS) is a nondestructive bioanalytical technique with a high signal-to-noise ratio and high ultraspatial resolution (3-10 μm). It is capable to explore the microstructures of plant tissues in a chemical sense and provide information on the composition, structure, and distribution of chemical compounds/functional groups. The objective of this study was to illustrate how SR-IMS can be used to image the internal microstructures of chickpea seed tissue within a cellular level. Chickpea seeds (CDC Cory) were collected from the Crop Development Center (University of Saskatchewan, Saskatoon, SK). The seeds were frozen at -20 °C on object disks in a cryostatic microtome and then were cut into thin cross sections (ca. 8 μm thick). The experiment was carried out on the mid-infrared beamline (01B1-1) at the Canadian Light Source (Saskatoon, SK). We obtained the ultraspatial images of the chickpea tissue with pixel-sized increments of imaging steps. The results showed that, with the extremely bright synchrotron light, spectra with high signal-to-noise ratios can be obtained from an area as small as 3.3 μm × 3.3 μm, allowing us to observe the seed tissue within a cellular level. Chemical distribution of chickpea such as lipids, protein, and carbohydrates could be mapped, revealing the chemical information of the chickpea internal microstructure. In conclusion, SR-IMS can rapidly characterize the molecular structure of protein, carbohydrates, and lipids at an ultraspatial resolution.

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.

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.

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.

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.

Three-dimensional (3D) representation of whole-brain cellular connectomics is the fundamental challenge for brain-inspired intelligence. And orderly automatic collection of brain sections on the silicon substrate is essential for the 3D imaging of cerebral ultrastructure. With the self-designed automated silicon-substrate ultra-microtome, serial brain sections can be orderly collected on the circular silicon substrates. In order to automate the collection process and further improve the efficiency of section collection, the form-invariant “Single Shot MultiBox-Detector” is proposed to detect the brain sections and baffles in the field of view of the microscope. And the “Cycle Generative Adversarial Networks” data augmentation method is proposed to alleviate the problem of fewer samples of the collected microscopic image dataset. The experimental results suggest that the proposed detection method could effectively detect the foreground objects in the microscopic images.

This study aimed to investigate the anatomical structure and fiber quality of four lesser-used wood species namely Benuang (O. sumatrana), Duabanga (D. moluccana), Pisang Merah (H. hellwigii), and Terap (A. odoratissimus). This study evaluated its suitability for raw material in pulp and paper manufacturing. The anatomical structure was observed macro- and microscopically. Macroscopic structures were observed directly to the wood samples, while microscopic characteristics were observed through microtome specimens. Fiber dimension was measured through macerated specimens and fiber quality was analyzed following the Rachman and Siagian’s method. Results showed that these four timber species have similarity in the indistinct growth ring, diffuse porous in a radial pattern, rounded solitary vessel outline, 1 to 3 cells of ray width, deposits within the rays, fiber length, and cell wall thickness. Differences were found on vessel diameter, vessel grouping, vessel frequency, tyloses existence, type of axial parenchyma, and ray height. Based on fiber length and its derived values, the wood fibers of all species studied are suitable for pulp and paper manufacturing. They belong to the II quality class. The produced pulp and paper would have good quality, especially in tensile, folding, and tear strength. To promote their utilization, silviculture aspect of these four species has to be well understood.

目 的：ミクロトーム連結式パラフィンブロック固定台冷却装置による薄切時持続冷却（持続冷却）と薄切された標本の厚さ（標本厚）評価におけるデジタル画像解析（DIA）の有用性を検討した．方 法：魚肉ソーセージのホルマリン固定パラフィン包埋ブロックを持続冷却し薄切した場合と保冷剤での事前冷却後薄切した場合（保冷剤冷却）について，エオシン染色後DIAでエオシン陽性面積率を比較検討した．結 果：設定値2～8μmで薄切した時の陽性面積率は対数曲線を示し，高い相関係数（R2>0.93）を示したため検量線として用いた．次に3μmの設定値で3名が各20枚薄切した平均値を比較すると，持続冷却は4μm以下，保冷剤冷却では6μm以上であった．結 論：DIA技術を用いると標本厚の推定が可能であり，保冷剤冷却ではブロックの膨張で設定値の約2倍の厚みの標本が作製されている可能性が示された．

目 的：ミクロトーム連結式パラフィンブロック固定台冷却装置を用いたパラフィンブロックの薄切時連続冷却の有用性を検討した． 方 法：検体のない疑似試料をパラフィンのみで作製し，冷却装置使用時と未使用時のブロック表面温度（温度）測定と連続薄切可能な厚みを検討した．また，その際ブロック裏面の隙間を充填するための義歯安定剤の検討も併せて行った． 結 果：冷却装置を用いると，義歯安定剤充填により一種類を除いてブロック温度は，開始時と 5 分後，開始時と 20 分 後の差が充填なしと比較して有意に大きかったが，20 分後のブロック温度に充填剤の有無で有意差はなかった．また，義歯安定剤充填・冷却条件での薄切は，1 μm 厚まで失敗することなく連続薄切でき，未冷却では，2 μm 厚まで連続薄切できた． 結 論：パラフィンブロックの薄切時持続冷却によって安定した連続薄切が可能であった．

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.

In the fabric industry, textile yarns are the fundamental building blocks. Hence, visualizing and studying yarn structure is essential to understand the structure and behavior of the fibers. Obtaining the yarn’s cross-section images is crucial in the calculations of yarn’s porosity; furthermore, a more precise expansion for the fiber’s migration can be concluded from the cross-sectional images. In this paper, three different methods (microtome, micro-computed tomography, and epoxy grinding–polishing methods) to image and visualize the yarn’s cross-section are presented. The experimental techniques are compared in terms of result useability, time of preparation, and overall outcome of the cross-sectional image. The images can be used for fiber distribution, air gap calculation, and twist analysis as well. The fiber diameter distribution of polyester yarn was measured based on the images obtained by the three different methods; the average fiber diameter measured based on the combined data from the three different methods was found to be 10.90 ± 0.30 µm. Published version

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

Summary Here, we describe a detailed workflow for ATUM-FIB microscopy, a hybrid method that combines serial-sectioning scanning electron microscopy (SEM) with focused ion beam SEM (FIB-SEM). This detailed protocol is optimized for mouse cortex samples. The main processing steps include the generation of semi-thick sections from sequentially cured resin blocks using a heated microtomy approach. We demonstrate the different imaging modalities, including serial light and electron microscopy for target recognition and FIB-SEM for isotropic imaging of regions of interest. For complete details on the use and execution of this protocol, please refer to Kislinger et al. (2020)., Graphical abstract, Highlights • A protocol for serial semi-thick ultramicrotomy of resin-embedded neuronal tissue • Serial semi-thick sections on tape inspected by light and scanning electron microscopy • Targeting regions of interest by FIB-SEM with high resolution isotropic voxels, Here, we describe a detailed workflow for ATUM-FIB microscopy, a hybrid method that combines serial-sectioning scanning electron microscopy (SEM) with focused ion beam SEM (FIB-SEM). This detailed protocol is optimized for mouse cortex samples. The main processing steps include the generation of semi-thick sections from sequentially cured resin blocks using a heated microtomy approach. We demonstrate the different imaging modalities, including serial light and electron microscopy for target recognition and FIB-SEM for isotropic imaging of regions of interest.

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.

Three-dimensional (3D) cellular scale imaging techniques that improve our understanding of the brain architecture is crucial for structural and functional integration and analysis of normal and pathological conditions in the brain. We have developed a wide-field fluorescent microscope using deep ultra violet (DUV) light emitting diode as the illumination source. This microscope employs oblique illumination of deep UV light and the optical sectioning is obtained on the tissue surface over a few micron thickness; largely attributed to the large absorption and hence low tissue penetration of the DUV light. Fluorescence emissions in the visible range that are spectrally separates allows for multiple channel of fluorophore detection using single or a combination of dyes. The fluorescence signal is captured using water immersion objectives and a color camera. Arduino Mega 2560 controlled 3-axis motorised microscope stage is developed for obtaining the wide-field imaging. To enable 3D imaging, the microscope setup is integrated with vibrating microtome that can slice thin sections for serial block-face imaging. In this paper, we show the versatility of the DUV microscope as a microscopy tool for use in neuroscience labs for a range of 2D brain imaging applications. We also present its applicability for 3D imaging of rodent brain in combination with whole brain staining protocol.

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.

With the wide application of ultra-microtome sectioning in the preparation of transmission electron microscopy (TEM) specimens with bio- and organic materials, here, we report an ultra-microtome-based method for the preparation of TEM specimens from cathodes of Li-ion batteries. The ultra-microtome sectioning reduces the sample thickness to tens of nanometers and yields atomic resolution from the core region of particles of hundreds of nanometers. Analysis indicates that the mechanical cross-sectioning introduces no observable microstructural artifacts or structural damage, such as microcracking and nanoporosity. These results demonstrate the high efficiency of the ultra-microtome approach in preparing well-thinned specimens of particulate materials that allow for atomic-scale TEM imaging of a large number of sectioned particles in one single TEM specimen, thereby providing statistically significant results of the TEM analysis.

Three-dimensional (3D) cell cultures are rapidly emerging as a promising tool to model various human physiologies and pathologies by closely recapitulating key characteristics and functions of in vivo microenvironment. While high-throughput 3D culture is readily available using multi-well plates, assessing the internal microstructure of 3D cell culture models yet remains extremely slow because of manual, laborious and time-consuming histological procedures. Here we present a four-dimensional (4D) printed transformable tube array (TTA) using a shape memory polymer (SMP) to enable massively parallel histological analysis of 3D cultures. The interconnected TTA can be programmed to be expanded by 3.6 times of its printed dimension to match the size of a multi-well plate, with the ability to restore its original dimension for transferring all cultures to a histology cassette in order. Being compatible with microtome sectioning, the TTA allows for parallel histology processing for the entire samples cultured in a multi-well plate. The test result with human neural progenitor cell (hNPC) spheroids suggests a remarkable reduction in histology processing time by an order of magnitude. High-throughput analysis of 3D cultures enabled by our TTA has great potential to further accelerate innovations in various 3D culture applications such as high-throughput/content screening, drug discovery, disease modeling, and personalized medicine.

Helianthus annuus L. as an oil seed crop is widely grown throughout the world. One of the most destructive diseases of sunflower is stem rot caused by Sclerotinia sclerotiorum. Oxalic acid is the major virulence factor of this necrotrophic pathogen. It is important to further investigate plant responses to this non-specific toxin. The basal stems of sunflower partially resistant and susceptible lines were treated with 40 mM OA (pH 3.7) of fungus culture filtrate and samples were collected at 24, 48 and 72 hours post treatment. SDS-PAGE protein pattern and total soluble protein were determined in both lines after treatment. In SDS-PAGE protein pattern new protein bands appeared in both lines after treatment. These observations suggest induction of stress-related proteins upon culture filtrate treatment. The identities of these new proteins need to be more clarify in future investigations. The changes in xylem morphology and degree of lignification of both lines was studied by light microscopy and microtome sectioning techniques after treatment with S. sclerotiorum culture filtrate. Anatomical investigations revealed changes in xylem diameter and xylem lignification of treated lines at various time points. More lignin deposition in xylem vessels of partially resistant line has been observed after treatment. In addition, the size of xylem vessels in partially resistant line has been sharply decreased upon pathogen filtrate treatment. The results of this study will help us gain a more complete understanding of resistance mechanisms to this cosmopolitan and devastating pathogen.

Quantitative metabolomics places high demands on sample preparation, including a high degree of metabolite extraction and controlled sample weight. In respect to elastic collagen-rich tissues, the existing methods of sample homogenization poorly fit these demands due to incomplete homogenization, sample material loss, or metabolite degradation. Herein, a novel method based on the use of a microtome-cryostat apparatus is proposed. The performance of the cryotome method is compared with the results obtained with the use of a vortex bead beating. NMR-based metabolomic analysis shows that the extraction efficiency and the data scattering for both methods of sample preparation are similar. However, the heat generation during the bead beating causes the destruction of thermally-unstable compounds; besides, it may cause protein hydrolysis, leading to an artificial increase in the amino acid level. The cryotome method of sample homogenization does not cause sample heating, and it seems to be ideal for elastic tissues.

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.

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 leaf anatomy has been shown as an important tool to understand the interaction between this organ and its microclimate but this topic is still unclear for citrus plants. The objective of this study was to analyze the leaf structure of Ponkan, Fremont and Thomas tangerine and Murcott tangor. Fully expanded leaves were collected collected, from the 2nd and 3rd nodes of plagiotropic branches facing West, at three crown heights (lower, middle and upper). The leaves were fixed in 70% ethanol and embedded in historesin and sectioned in a semi-automatic microtome. The sections were stained with toluidine blue; images were captured under a light microscope coupled and analyzed to measure leaf tissue thickness. The Fremont tangerine leaves had greater epidermis thickness on the adaxial and abaxial surfaces and secretory cavity diameter as well as a shorter distance between secretory cavities, whereas the Murcott leaves had greater palisade and spongy parenchyma thickness. Thus, Fremont and Murcott are potential varieties for xeric conditions, as they are able to reduce transpiration and make optimal use of high incident radiation. The microclimates created by plant architecture may affect the leaf anatomy of the Ponkan, Fremont and Thomas and Murcott trees, allowing anatomical plasticity.