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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 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.

Understanding the structure and working principle of brain neural networks requires three-dimensional reconstruction of brain tissue samples using array tomography method. In order to improve the reconstruction performance, the sequence of brain sections should be collected with silicon wafers for subsequent electron microscopic imaging. However, the current collection of brain sections based on silicon substrate involve mainly manual collection, which requires the involvement of automation techniques to increase collection efficiency. This paper presents the design of an automatic collection device for brain sections. First, a novel mechanism based on circular silicon substrates is proposed for collection of brain sections; second, an automatic collection system based on microscopic object detection and feedback control strategy is proposed. Experimental results verify the function of the proposed collection device. Three objects (brain section, left baffle, right baffle) can be detected from microscopic images by the proposed detection method. Collection efficiency can be further improved with position feedback of brain sections well. It has been experimentally verified that the proposed device can well fulfill the task of automatic collection of brain sections. With the help of the proposed automatic collection device, human operators can be partially liberated from the tedious manual collection process and collection efficiency can be improved.

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.

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.

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.

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.

The use of unbiased stereology to quantify structural parameters such as mean cell and nuclear size (area and volume) can be useful for a wide variety of biological studies. Here we propose a novel segmentation framework using an Active Contour Model to automate the collection of stereology from stained cells and other objects in tissue sections. This approach is demonstrated for stained brain sections from young adult Fischer 344 rats. Animals were perfused in-vivo with 4% paraformaldehyde and sectioned by frozen microtomy at an instrument setting of 40 μm. For each rat brain, a systematic-random set of sections through the entire substantia nigra pars compacta (SN) were immunostained to reveal tyrosine hydroxylase (TH)-immunopositive neurons. The novel framework applied an active contour (modified balloon snake) model with non-constant balloon force to automatically segment and quantify neuronal cell bodies by stereological point counting (SPC). Several contours were initialized in the image and based on the contour fit after 200 iterations classified as immunopositive (signal) or background contours in a sequential manner. Cell contours were determined in four steps based on several criteria, e.g., area of contour, dispersion measure, and degree of overlap. The image was automatically segmented according to the final contours. Using a point grid automatically generated at systematic-random orientations over the images, points hitting the segmented neural cell bodies were automatically counted. The final values from the automatic framework were compared with findings for ground truth (manual SPC). The results of this study show a strong agreement between data collected by the automatic framework and the ground truth (R2 ≥ 0.95) with a 5× gain in time efficiency for the automatic SPC. These findings give strong support for future applications of pattern recognition for assessing stereological parameters of biological objects identified by high signal:noise stains.

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.

Microfibers are one of the most abundant microplastic particle types found in the environment, where they cause negative impacts on organisms and possibly on human health. Microfibers should be included in a wide range of laboratory studies; however, microfibers for scientific studies are not commercially available. Current methods to make microfibers generally create particles with large size ranges and poor precision, and efficient production of particles ≤100 µm is difficult. Laboratory studies of the biological and toxicological effects and chemical interactions of microfibers require uniform, small microfibers in sufficient numbers for environmentally relevant experiments. We developed a novel fiber embedding technique and modified a seminal cryomicrotomy method to produce precise microfibers in quantities suitable for environmentally relevant concentrations. Polyethylene terephthalate (PET) and nylon fibers were strategically wound onto a spindle, embedded in paraffin wax, and sectioned using a standard paraffin microtome. After processing with a suitable organic solvent to remove the wax, microfiber size distributions were assessed. The small microfibers (10-42 µm) were accurate to the target lengths with excellent precision and a production rate ≥13.5 times higher than previous methods. As a proof of application, three lengths of manufactured PET fibers were stained with Nile red and exposed to eastern oyster larvae (Crassostrea virginica) for 24 h. Larvae ingested the smaller fiber lengths (14 and 28 µm), and the Nile red-stained fibers were visible and distinguishable in the guts of the larvae. This experiment was the first to demonstrate ingestion of plastic particles other than microspheres by oyster larvae. The present method facilitates the use of small microfibers in laboratory experiments, allowing for a more complete understanding of microplastic effects in the environment. Environ Toxicol Chem 2022;41:944-953. © 2021 SETAC.

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.

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.

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.

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.

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

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 μm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy.

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.

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.

Background The aim of the authors was to examine the abutment-fixture interface in Morse-type conical implants in order to verify gaps at this level using a new microscopical approach. Material and Methods In this in vitro study, 20 abutment-fixture complexes were prepared by sectioning (longitudinal and cross-sectional to the long axis) with a microtome and then with a focused ion beam (FIB). This is a micrometric machine tool that uses gallium ions to abrade circumscribed areas to dig deeper into the cuts obtained with the microtome in order to eliminate cut-induced artifacts. This is because the FIB abrasion is practically free from artifacts, which are normally generated by the action of the microtome blades or other techniques. Samples were then observed by scanning electron microscopy (SEM). Results The observation of the abraded parts with the FIB permitted measurement of the real gap between the implant-abutment components. A variable amount of gap was retrieved (from 0 to 3 μm) by the observations, confirming the non-hermetic nature of the connection. It has to be pointed out that in approximately 65% of cases, the gap accounted for less than 1 μm. Conclusions The reported data confirmed that the analyzed connection system allowed for minimal gap. However, from the evidence of the present analysis, it cannot be assumed that the 2 parts of a Morse-type conical implant are fused in 1 piece, which would create a perfectly matched hermetic connection. Key words:Dental abutment, dental implant, focused ion beam SEM, implant-abutment connection, morse taper, microbial leakage.

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.

This method details our microtomy (sectioning) process for reasearch specimens involved with HuBMAP. This process follows the microtomy process that follows after receiving, processing, and embedding of research specimens into formalin fixed, paraffin embedded tissue blocks.

Background Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory. Design After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining. Results Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2–3 days. The incremental cost over that of routine histology is nominal. Conclusion This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically.

Background Traditional sutures, staples and surgical tapes are the most common methods used to close wounds in veterinary patients. However, these methods can induce granuloma and fistulas formation, loose sutures and wound leaks. Lately, tissue adhesives such as cyanoacrylates (Histoacryl®) have become popular, this glue can bond tissues such as endothelium, mucosa, skin, blood and bone rapidly. Studies using cyanoacrylates have shown bond strength, bacteriostatic and hemostatic properties. The objective of this work is to compare the use of n-butyl-cyanoacrylate with non-absorbable sutures in tissue regeneration by histology and microbiological analysis in mice. (All procedures were approved by the USFQ Ethics Committee on Animal Experimentation, Doc# 2020-001). Methods Two cuts of 1 cm were perfomed in the dorsal skin of each mouse: one cut was bonded by simple suture (control), and the other with tissue adhesive (Histoacryl). Albino mice were anesthetized before the surgical procedure; analgesics were provided in the water after the surgery to minimize pain. After 48 hours of the surgical procedure, the mice were euthanized and biopsies of 2 cm2 were collected for each cut. Three mice were used for histological evaluation and two for microbiology tests. Assays were repeated 4 times. For microbiology tests, samples were resuspended in 1 ml of TBS culture broth by vortex. Colony forming units (CFU) were evaluated for two dilutions after incubation on Nutrient Agar for 48 hours. For histological analysis, biopsies were fixed with formaldehyde, cut by a microtome (Leyca RM2155) and stained with trichrome, haematoxylin and eosin. Statistical analysis was performed by Mann-Whitney and Kruskal-Wallis tests using GraphPad Prism software. Results No differences were found in the microbiological and histological analysis. Conclusions The results showed that there is no significant diference between n-butyl-cyanoacrylate and non-absorbable sutures, no increased risk of microbiological contamination or regenerative improvements were shown.

Objectives: This study has been conducted to unveil adhesion promoter's underlying microscopic mechanism using confocal microscope. Materials and Methods: Fifty maxillary first premolars (25 fluorosed and 25 nonfluorosed) were selected. The teeth were divided into 4 groups of 10 teeth each based on the adhesion booster applied and two control groups of 5 fluorosed and 5 nonfluorosed teeth without adhesion booster. Two different adhesion boosters, All-Bond 3 (Bisco, Schaumburg, IL, USA) and Enhance LC (Reliance, Itasca, IL, USA), were mixed with Rhodamine B fluorescent dye and applied on the tooth to facilitate the detection of resin tags in the confocal microscope. The teeth were sliced to a cross-section of 800 μm by hard tissue microtome. Each cross section was scanned in confocal microscope till three distinct resin tags were seen, and this image was stored and quantified. The depth of penetration of adhesive resin tag was chosen as a measure of micromechanical bonding. Data obtained were statistically analyzed. Results: The maximum depth of penetration was recorded in fluorosed/All-Bond 3 group of 75.57 μm and a minimum depth of penetration was in fluorosed and conventional primer group of 26.46 μm. Conclusions: This shows that micromechanical bonding is compromised in fluorosed teeth. Both the adhesion boosters, All-Bond 3 and Enhance LC, enhanced the depth of resin penetration in fluorosed as well as nonfluorosed teeth while conventional primer did not prove to be very useful. Thus, adhesion boosters have been beneficial to increase the micromechanical bonding in cases where bonding is compromised, thus providing a good solution to effective orthodontic bonding.

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post-fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512-517, 2016. © 2016 Wiley Periodicals, Inc.

Objective: To analyze mast cell histomorphometry in three different regions of the human intracranial dura mater. Method: Three specimens of dura mater were collected after approval by the Ethics Committee (CAAE No. 57692216.5.0000.5208). Each dura mater was obtained from human cadavers between 7 and 24 hours after death. After collection, the samples were fixed, cut into two fragments and longitudinally placed in the following way: external (periosteum) and internal (meningeal) sides. The fragments (1.5 cm2) were taken from three different regions: proximity of the right middle meningeal artery, the proximity of the left middle meningeal artery and superior sagittal sinus. These fragments were submitted to microtomy (10 μm), stained with 0.1% toluidineblue and analyzed by optical microscopy. The histomorphometric parameters adopted were: the distance from the mast cells to the vessels, the number and if the mast cells were degranulated. Five fields from each case were analyzed. For this analysis, the Image J 1.52a 2019 software was used. Results: A higher number of mast cells was observed in the periosteal layer when compared withthe meningeal layer (p=0.026). When the distribution of the mast cells was evaluated, we observed that the cells were localized in the proximity of the middle meningeal artery (p

[EN] The objective of this work was to establish a three-dimensional measuring method for the size, morphology and distribution of internal structure such as ice crystals, bubbles and solids content within an ice cream sample by using a cryogenic microtome spectral imaging system (CMtSIS). The 3-D images of ice crystals, bubbles and milk solids were recognized by reconstructing the circles in 2-D images into 3-D spheres; and the Overrun by Volume (ORV) was obtained by incorporating the area of bubbles on integrated image and the volume of bubbles in the 3-D image., This work was supported by a Kakenhi Grant-in-Aid (No. 17K00826) from the Japan Society for the Promotion of Science (JSPS).

We developed a computational framework for automated integration of a large number of two-dimensional (2D) images with three-dimensional (3D) image datasets located in the standard 3D coordinate. We applied the framework to 2,810 para-sagittal sectioned mouse brain 2D images of in situ hybridization (ISH), archived in the BrainTx database (http://www.cdtdb.neuroinf.jp). We registered the ISH images into the mouse standard coordinate space for MR images, Waxholm space (WHS, https://www.nitrc.org/projects/incfwhsmouse) by linearly transforming them into each of a series of para-sagittal MR image slices, and identifying the best-fit slice by calculating the similarity metric value (δ). Transformed 2D images were compared with 3D gene expression image datasets, which were made using a microtomy-based microarray assay system, Transcriptome Tomography, and archived in the ViBrism DB (http://vibrism.neuroinf.jp): the 3D images are located in the WHS.We first transformed ISH images of 10 regionally expressed genes and compared them to signals of corresponding 3D expression images in ViBrism DB for evaluating the integration schema: two types of data, produced with different modalities and originally located in different dimensions, were successfully compared after enhancing ISH signals against background noise. Then, for the massive transformation of BrainTx database images, we parallelized our framework, using the IPython cluster package, and implemented it on the PC cluster provided for the Brain Atlasing Hackathon activity hosted by Neuroinformatics Japan Center in Japan. We could identify the best-fit positions for all of the ISH images. All programs were made available through the GitHub repository, at the web site of neuroinformatics/bah2016_registration (https://github.com/neuroinformatics/bah2016_registration).

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Aim. This study sought to compare the microleakage of three adhesive systems in the context of Erbium-YAG laser and diamond bur cavity procedures. Cavities were restored with composite resin. Materials and Methods. Standardized Class V cavities were performed in 72 extracted human teeth by means of diamond burs or Er-YAG laser. The samples were randomly divided into six groups of 12, testing three adhesive systems (Clearfil s3 Bond Plus, Xeno® Select, and Futurabond U) for each method used. Cavities were restored with composite resin before thermocycling (methylene blue 2%, 24 h). The slices were prepared using a microtome. Optical microscope photography was employed to measure the penetration. Results. No statistically significant differences in microleakage were found in the use of bur or laser, nor between adhesive systems. Only statistically significant values were observed comparing enamel with cervical walls (p < 0.001). Conclusion. It can be concluded that the Er:YAG laser is as efficient as diamond bur concerning microleakage values in adhesive restoration procedures, thus constituting an alternative tool for tooth preparation., SCOPUS: ar.j, info:eu-repo/semantics/published

In neuroscience, the optical fractionator technique is frequently used for unbiased cell number estimations. Although unbiased in theory, the practical application of the technique is often biased by the necessity of introducing a guard zone at one side of the disector to counter lost caps and/or optical limitations. Restricting the disector within the section thickness potentially introduces bias in two ways. First, the need to measure section thickness in order to obtain the disector height/section thickness fraction is challenging since both microcator measurements, microtome block advance, and measurements on re-embedded sections are potentially biased. Second, disector placement is not uniform random within the section thickness resulting in a bias in most sections with inhomogeneous cell distribution along the z axis. Re-embedded 2-hydroxyethylmethacrylate (hereafter methacrylate) sections were inspected for lost caps to evaluate the possibility of whole section thickness counting with the optical fractionator technique and hippocampal granular cell nucleoli density differences along the z axis were assessed with a z axis analysis. No lost caps were found in the examined re-embedded tissue and an inhomogeneous cell distribution through the section thickness was observed. In thick methacrylate sections devoid of lost caps sampling through the entire section thickness could be an acceptable alternative to the use of guard zones and the consequent biases associated with section thickness measurement and non-random placement of disectors.

Background: Type II microcalcifications, most often seen in proliferative lesions, including both benign and malignant pathologies. But whether the emergence of type II microcalcifications associated with cell canceration is still not clear now. Raman spectroscopy is a powerful, non-invasive analytical tool which can provide detailed and meaningful information about biochemical composition of tissues at molecular level. Our aim was to find the differences and relationships of type II microcalcifications between fibroadenoma, ADH tissues, and DCIS, IDC in breast based on their various biochemical characteristics by Raman spectroscopy. Methods: The frozen sections were collected from 15 patients (all female; ages 25-57) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20∼-25° and two contiguous sections (6 µm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis and found the microcalcification locations by three expert breast pathologists; the same position of the other section was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the H&E section). After the spectra we needed had been obtained, the Au@SiO2 shell-isolated nanoparticles (SHINs) were added to the surface of frozen sections immediately and then the spectra with SHINs were collected. Results: A total of 122 Raman spectra and 119 SHINERS spectra were obtained from the microcalcifications in 3 fibroadenoma tissues, 3 ADH, and 5 DCIS, 4 IDC breast tissues. The Raman signals were significantly enhanced by SHINs. Except the major peak at 958-960 cm-1 which attributed to the vibrational modes of calcium hydroxyapatite, the results show no calcium oxalate dihydrate peaks but several other chemical species. The peaks of these species appear at 1002, 1072, 1126, 1446, 1556, and 1657 cm-1, these are attributed to amino acid residue (phenylalanine), nucleic acids, lipids, carotenoids, and Amide I, respectively. The fibroadenoma and ADH microcalcifications mean spectral have the same peak at 1072cm-1, which belongs to the O-P-O stretch of nucleic acids, but in DCIS and IDC tissues the peak changes to 1078cm-1. The ADH have more obvious peak at 1657 cm-1 which assigned to different vibrational modes of the backbone and Amide I, but in DCIS and IDC tissues, the Amide I bands were disappeared. In the DCIS microcalcifications mean spectral, the peaks attributed to amino acid residue (phenylalanine) at 1003, 1031 cm-1 and lipids (CH2 and CH3 bending) at 1301, 1441 cm-1 show more stronger. Meanwhile, compare with DCIS, the IDC spectral shows more stronger nucleic acids peaks and weaker lipids peaks. Conclusion: We have demonstrated the potential of SHINERS to differentiate Type II microcalcifications found in fibroadenoma, ADH, and DCIS, IDC breast tissues. The results presented in this paper provide the biochemical characteristics of the Type II microcalcifications among these tissues, and may represent a key factor responsible for mechanisms of carcinogenesis. Citation Format: Haipeng Zhang, Bing Han, Chao Zheng, Ye Du, Lijia Liang, Weiqing Xu, Zhimin Fan. Exploring type II microcalcifications in benign, premalignant and malignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-15.

Background: Atypical ductal hyperplasia (ADH) is a premalignant breast lesion associated with an increased risk of breast cancer which shares some but not all the features of ductal carcinoma in situ (DCIS). The mechanism that ADH changes to DCIS and even invasive ductal carcinoma (IDC) is still not clear now. There is a pressing need to develop screening techniques that are accurate, minimally invasive and, more sensitive to identify early neoplastic changes and hence improve early breast cancer diagnosis. Our aim was to explore the differences and relationships between normal, ADH, DCIS and IDC lesions of the breast based on biochemical characteristics determined by Raman spectroscopy Methods: Frozen sections were collected from 39 patients (all ladies) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20-25 ° and two contiguous sections (6 µm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis; the same position of the other section was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the H&E section). After Raman measurement, the sections were routinely processed, stained with H&E and histologically examined. Support vector machine (SVM) was used to differentiate different breast lesions. Results: A total of 475 Raman spectra were obtained from 9 normal breast tissues, 7 ADH, 8 DCIS, and 15 IDC breast tissues. Pronounced mean Raman spectra differences were observed between normal tissues, ADH, DCIS and IDC tissues. The significant features of normal tissues are 1301, 1438, 1652, and 1743 cm-1, these peak positions are attributed to lipids, and the spectra profiles of normal tissues have no strong protein peaks. Most noticeable was the increased protein and reduced lipid levels of ADH tissues compared to normal tissues. The peak relative intensity of 1158 cm-1 which is attributed to the vibrational modes of C-C stretch of proteins has significantly increased from normal tissues to DCIS and IDC in breast. The major spectra differences in ADH, DCIS and IDC spectra were evidenced by a red shift with a broad peak of CH2, the intensity of the stretching vibration peak of carotenoids, a relatively strong band of amide-I, and the nuclear acid peak. ADH tissues had the largest constituent variations between subjects. During the disease progression, IDC tissues have smaller inter-subject constituent variations than DCIS and ADH tissues. The accuracies of SVM are 93% and 95% in discriminating normal and IDC, while only 50% and 51% in discriminating ADH and DCIS. Conclusion: Malignant transformations in tissue are associated with complex biochemical changes and may provide an effective way to evaluate the malignancy by Raman spectroscopy as it reveals differences between normal tissues, ADH, DCIS and IDC tissues in the breast. Further study to explain the biochemical relationships between these differences will shed more light into a better understanding of the mechanism by which ADH and DCIS convert to IDC. Citation Format: Bing Han, Haipeng Zhang, Chao Zheng, Ye Du, Lijia Liang, Weiqing Xu, Zhimin Fan. Explore the differences and relationships between normal and ADH, DCIS and IDC tissues in breast based on Raman spectroscopy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-10.

Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.

An acoustic imaging microtome system (AIMS) was constructed to map the internal structure of fish. The system consists of two pairs of high-frequency (4.5 MHz) transmit-and-receive planar arrays, with one pair in the vertical plane and the other in the horizontal plane. AIMS provides a series of acoustic images, analogous to microtome slices, along the length of a fish by combining electronically controlled sequential acoustic transmissions and receptions over 224 channels and a computer-controlled mechanical device that moves the acoustic arrays lengthwise along the fish. AIMS measures the acoustic attenuation (extinction) along the direct ray paths of each transmit-receive channel pair that penetrates the fish body in a bistatic configuration. This results in a 2-D image of the interior morphology with 4- to 5-mm resolution. The images can provide morphological information on the fish's cheekbone, backbone, rib bone, skull, fins, stomach, and swimbladder, which is important for accurately modeling fish acoustic target strength, especially for non-swimbladder-bearing fish species.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

Despite the recent advent of neuro-radiographic techniques, creating a ‘perfect’ human brain atlas providing precise and consistent images with minimal distortion is practically difficult. In this study, we created a new human brain atlas from cadaveric brains with serial sections of 50 μm thickness covering the entire basal ganglia. Human cerebral hemispheres were obtained from 10 donated cadavers and fixed in 10% formalin solution, cut in a block measuring 50 mm × 30 mm × 50 mm around the midpoint of the anterior and posterior commissures and frozen at −40 °C. Each block was cut into 50-μm-thick sections on the freezing microtome and the cross-sectioned surface was photographed. Simultaneously, every 10th slice from one sagittal hemisphere was sampled and stained using the Kluver-Barrera method. Prepared slides were photographed under light microscopy, and data from digital images of the cross-sectioned surface (DICSS) and digital images from microscopic sections (DIMS) were processed. Gray areas on DICSS largely represented areas of dense cellularity, and around subthalamic nucleus (STN), the zona incerta and field of Forel were clearly distinguishable on the anterosuperior side, as was the substantia nigra on the caudal side. DICSS successfully delineated the anatomical structure identical to the STN and surrounding contiguous nuclei. This new brain atlas will allow elucidation of anatomy that cannot be clearly disclosed from modern radiographic imaging or is very difficult to analyze with spatially inconsistent histological sections, and will contribute to further progress in anatomical studies of the human basal ganglia.

We present SBEMimage, an open-source Python-based application to operate serial block-face electron microscopy (SBEM) systems. SBEMimage is designed for complex, challenging acquisition tasks, such as large-scale volume imaging of neuronal tissue or other biological ultrastructure. Advanced monitoring, process control, and error handling capabilities improve reliability, speed, and quality of acquisitions. Debris detection, autofocus, real-time image inspection, and various other quality control features minimize the risk of data loss during long-term acquisitions. Adaptive tile selection allows for efficient imaging of large tissue volumes of arbitrary shape. The software’s graphical user interface is optimized for remote operation. In its user-friendly viewport, tile grids covering the region of interest to be acquired are overlaid on previously acquired overview images of the sample surface. Images from other sources, e.g., light microscopes, can be imported and superimposed. SBEMimage complements existing DigitalMicrograph (Gatan Microscopy Suite) installations on 3View systems but permits higher acquisition rates by interacting directly with the microscope’s control software. Its modular architecture and the use of Python/PyQt make SBEMimage highly customizable and extensible, which allows for fast prototyping and will permit adaptation to a wide range of SBEM systems and applications.

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.

Objectives To develop an economic, practical and readily available animal model for preclinical testing of urethral bulking therapies, as well as to establish feasible experimental methods that allow for complete analysis of hard microparticle bulking agents. Methods Alumina ceramic beads suspended in hyaluronic acid were injected into the proximal urethra of 15 female rats under an operating microscope. We assessed overall lower urinary tract function, bulking material intraurethral integrity and local host tissue response over time. Microphotographs were taken during injection and again 6 months postoperatively, before urethral harvest. Urinary flow rate and voiding frequency were assessed before and after injection. At 6 months, the urethra was removed and embedded in resin. Hard tissue sections were cut using a sawing microtome, and processed for histological analysis using scanning electron microscopy, light microscopy and immunohistochemistry. Results Microphotographs of the urethra showed complete volume retention of the bulking agent at 6 months. There was no significant difference between average urinary frequency and mean urinary flow rate at 1 and 3 months postinjection as compared with baseline. Scanning electron microscopy proved suitable for evaluation of microparticle size and integrity, as well as local tissue remodeling. Light microscopy and immunohistochemistry allowed for evaluation of an inflammatory host tissue reaction to the bulking agent. Conclusions The microsurgical injection technique, in vivo physiology and novel hard tissue processing for histology, described in the present study, will allow for future comprehensive preclinical testing of urethral bulking therapy agents containing microparticles made of a hard material.

T his protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-mm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitationcontraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in 4 h.

High-quality patient samples are required for reliable immunohistochemistry test outcomes that provide a significant benefit for patient care. Among the preanalytic variables in tissue handling, tissue thickness is thought to be easily controlled; however, whether the thickness of the tissue effects the staining intensity for antibody immunohistochemistry has not been quantitatively demonstrated. To investigate, we cut multiblock tissue sections of tonsil, liver, and kidney at 2, 4, 6, and 8 μm thicknesses. Interferometry measurements of the sectioned paraffin showed a