Sample Preparation for Electron Microscopy

Background: Generally, in practical dermatology, the term artifacts is used to describe what is called pathomimia which means self-inflicted lesions or that lesions induced accidentally on oral mucosa and skin. Regarding dermatopathology, the word, 'artefact' comes from the Latin term, 'Ars' = art and 'factum' = made. This study evaluates types of artifacts, frequencies, and their impact on final diagnosis. Patients and method: This observational, cross-sectional study. The study worked on 590 dermatological slides, that stained with hematoxylin and eosin stain. Slides evaluated from archive and that collected during time of study which occur from June 2022 to November 2022, in Al-Yarmouk Teaching Hospital, Baghdad, Iraq, analyzed for artifacts under light microscope. Results: Totally 590 slides were evaluated, only 69 (11.695%) show no artifacts. Slides with artifact(s) counting 521(88.305%). Histopathological artifacts were accounted for 931 because more 45% of total examined slides show two or more artifacts. Folding artifacts were the most common type, while artefacts related to fixation were the least 307(32.975%), 6(0.644%) respectively. Conclusion: Histopathological evaluation of skin biopsies had long, and multiple stages done by the dermatologist, assistant pathologists, and lab technicians. That led to increased incidence of errors and artifacts. Artifacts invariably occur in the majority of dermatopathological slides and may interfere with the interpretation of histopathological diagnosis. These artefacts should be recognized and do all effort to avoid or at least, minimized them, which yet defied even with soft tissue handle and wrong techniques avoidance.

In this paper, we present three cases studies with dedicated FIB-TEM technique that developed to characterize each of them. The first case describes a FIB sample preparation method that can include two columns of the structure of interest into one sample. It is a useful technique to characterize the overlay offset when more than one column of the structures is concerned. The second case is about to prepare an ultra-thin TEM sample which minimizes the shadowing effect and nano-roughness in localized area. Such thin samples are ideal for high resolution imaging and crystal structure analysis. In the last case, one TEM imaging technique is developed to enhance the defect contrast by utilizing the Fresnel fringes when there is not enough contrast can be obtained from normal in focus imaging condition. Theoretical description of the Fresnel fringes is also provided to illustrate the origin of extra contrast that can obtain from this technique.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

To contribute to a celebration marking the centennial of Otto Scherzer's birth is a pleasant but daunting honor. For the majority of the twentieth century, his insights dominated transmission electron microscopy. Not only did he provide the effective engineering constraints of all electron microscopes that attempted the highest spatial resolution but also sowed the seeds that lead to the lifting of those constraints. Practicable aberration correction was initiated by him and this paper will describe some recent applications.

While FIB sample preparation for transmission electron microscopy is a well established technique, few examples exist of samples of sufficient quality for atomic resolution imaging by aberration corrected (scanning) transmission electron microscopy (STEM). In this work we demonstrate the successful preparation of such samples from five different materials and present the refined lift-out preparation technique, which was applied here. Samples with parallel surfaces and a general thickness between 20 and 40 nm over a range of several mm were repeatedly prepared and analyzed by Cs-corrected STEM at 60 and 100 kV. Here, a novel 'wedge pre-milling' step helps to keep the protective surface layers intact during the whole milling process, allowing features close to or at the sample surface to be analyzed without preparation damage. Another example shows the cross-sectional preparation of a working thin film solar cell device to a final thickness of 10 to 20 nm over mm sized areas in the region of interest, enabling atomic resolution imaging and elemental mapping across general grain boundaries without projection artefacts. All sample preparation has been carried out in modern Dual-Beam FIB microscopes capable of low-kV Ga þ ion milling, but without additional preparation steps after the FIB lift-out procedure.

Bi 2 Se 3 is a 3D topological insulator (TI) that has attracted a lot of research interest due to its exotic properties [1], associated with topologically-protected helical two-dimensional surface states and onedimensional bulk states associated with line defects such as dislocations. Recent theoretical studies [2] have shown that when graphene is placed near Bi 2 Se 3 the strong spin orbit interaction due to proximity effects will open the band gap in graphene for 0.2 eV. Therefore TI/graphene heterostructures are promising platform for developing electronic and spintronic graphene based devices.

Inclusion criteria: Normal and well-fixed cervix, endometrium, ovaries, artery, smooth muscle, skeletal muscle and nerve bundles. Exclusion criteria: Biopsies that were not processed, not fixed properly and with inadequate tissue structure were not included, malignant neoplasms were not included.

This letter describes an innovative spin-coating system, built from off-the-shelf components, that can easily and inexpensively be integrated into any laboratory environment. Combined with a liquid suspension of conductive polymer, such a "rotary coater" enables simple coating of planar samples to create a physical protective barrier on the sample surface. This barrier aids in charge dissipation during scanning electron microscope and focused ion beam (FIB) imaging and provides wide-scale protection of the sample surface from ion bombardment during FIB imaging and gas-assisted deposition. This polymer layer replaces the localized and time-consuming electron beam deposition step typically performed during transmission electron microscopy lamella preparation. After observation, the coating can be easily removed, if desired. The described spin-coating procedure has minimal cost while providing repeatable positive results, without the need for expensive commercial coating inst...

Introduction: Stains are a crucial component of laboratory procedure. They help in highlighting the different tissues, both normal and abnormal, which plays an important role in diagnostic histopathology. Synthetic dyes are utilised for majority of the stains in histology. Among the natural surrogates studied, Kumkum is less researched. Aim: To compare the staining characteristics of Kumkum, which imparts red colour as an alternative to eosin. Materials and Methods: This observational study was conducted at Histopathology section, Pathology department at Chettinad Hospital and Research Institute for a period of six months from January 2022 to June 2022. In 50 tissue blocks, each was made into two sections and one was stained with routine Haematoxylin and Eosin (H&E), while the other was stained with Haematoxylin and Kumkum solution (H&K). The Cellular Architecture (CA) (based on distinct or indistinct nucleus, cytoplasm) and quality of staining (poor, satisfactory or good) of H&E an...

In ceramic thin films, choosing an appropriate sample preparation method for transmission electron microscopy (TEM) analyses is of paramount importance to avoid preparation-induced damage and retain nanoscale features that require investigation. Here we compare two methods of TEM thin film sample preparation, namely conventional wedge polishing and focused ion beam (FIB) based lift out preparation applied to ferroelectric barium titanate (BaTiO3, BT) thin films made by chemical solution deposition (CSD). The aim of the work is to determine the pros and cons of each method considering not only the quality of the TEM specimen, but also aspects such as availability, ease of use, and affordability. Besides some limitations on the selection of visualized area due to thickness constraints on the FIB-made sample, both methods offer the capability to prepare samples with very comparable quality, as indicated by achieving the same thickness, a largely agreeing microstructure, no secondary ph...

Giemsa stain is one of the synthetic stains consisting of eosin and blue methylene that are useful for staining cells. However, the use of synthetic stain turns out to have several drawbacks, some of which are not eco-friendly, quite expensive and harmful to health. Therefore, there was a need of alternative coloring that is natural, more eco-friendly, inexpensive, and especially not harmful to health. This study used alternative dye, jamblang ethanol extract (Syzigium cumini), containing anthocyanins. Anthocyanins are a class of organic chemical compounds that could dissolve in polar solvents, and were responsible for giving orange, red, purple, blue, and black colors in higher plants such as flowers, fruits, seeds, vegetables and tubers. The results of this study indicated that Giemsa stain contained eosin and blue methylene so that while in use, it could produce blood cells such as erythrocytes, leukocytes and platelets whose appearance was clear under a microscope. Meanwhile, jamblang ethanol extract stain contains acidic anthocyanins so that this staining can only stain blood cells in the form of erythrocytes, if the leukocytes and platelets could not be stained properly due to leukocytes and platelets having acidic cell nuclei. Therefore, it can be concluded that the staning of Giemsa has a better quality than that of jamblang ethanol extract, but jamblang extract can be used as an alternative stain from Giemsa to indicate erythrocyte cells.

Polymerase Chain Reaction 12 commensal? Studies have attempted to address this question 1 with guidelines formulated for practice, but these are indeed merely "guidelines," because only identification of a potential pathogen within a site of infection can provide substantive evidence that the fungus is an invasive pathogen. For this and other reasons to be addressed in this text, the pathologic diagnosis of infection is a critical element in formulating optimal therapy. Diagnosing Infection In Situ Because host immune mechanisms can greatly amplify the host response, the actual numbers of pathogens present in tissues is frequently surprisingly small. This means that many sections may need to be examined before a pathogen is identified. Although

Over the last decades, research on microbial bioerosion affecting archaeological bone assemblages highlighted the fact that this is a significant factor determining the long-term survival of vertebrate hard tissues as well as the quality of the biological information retrievable from them (e.g. isotopic, genetic, histomorphological). In spite of this, information about bioerosion is still scarce or inexistent for most regions around the world. Among the likely causes of this situation are the perceived and factual technical difficulties that surround the implementation of a standard research on this subject. Taking this into account, the aim of this paper is twofold: on the one hand, to describe a protocol for the preparation of bone samples (thick sections) suitable for observation with BSE-SEM that fulfils the criteria of simplicity, low cost and effectiveness; on the other hand, to present and discuss the first results derived from the application of such protocol to artiodactyl bone samples recovered at different Late Holocene archaeological sites from the southern Pampas of Argentina. The obtained results indicate that the implemented technique was effective in terms of providing good quality information at a very low cost as measured from the resources (time and materials) invested. In addition, the results show that a significant part of the analysed specimens exhibit extensive and intensive histological alteration compatible with the action of bacteria, which is unexpected in light of the currently prevailing model about the origin and conditions of the bacterial attack on animal bones in archaeological deposits (i.e. the so-called "endogenous model").

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Over the last decades, research on microbial bioerosion affecting archaeological bone assemblages highlighted the fact that this is a significant factor determining the long-term survival of vertebrate hard tissues as well as the quality of the biological information retrievable from them (e.g. isotopic, genetic, histomorphological). In spite of this, information about bioerosion is still scarce or inexistent for most regions around the world. Among the likely causes of this situation are the perceived and factual technical difficulties that surround the implementation of a standard research on this subject. Taking this into account, the aim of this paper is twofold: on the one hand, to describe a protocol for the preparation of bone samples (thick sections) suitable for observation with BSE-SEM that fulfils the criteria of simplicity, low cost and effectiveness; on the other hand, to present and discuss the first results derived from the application of such protocol to artiodactyl bone samples recovered at different Late Holocene archaeological sites from the southern Pampas of Argentina. The obtained results indicate that the implemented technique was effective in terms of providing good quality information at a very low cost as measured from the resources (time and materials) invested. In addition, the results show that a significant part of the analysed specimens exhibit extensive and intensive histological alteration compatible with the action of bacteria, which is unexpected in light of the currently prevailing model about the origin and conditions of the bacterial attack on animal bones in archaeological deposits (i.e. the so-called "endogenous model").

Dyes are the substances which are used for imparting color. They are mainly used in food, textiles, paint, leather & printing industries. Dyes are extensively used in histopathology and cytological examination in oral pathology. Hematoxylin and Eosin stains are primarily used for differentiating between cell nucleus and cytoplasm. However, Eosin been a xanthene derivative is synthetic in nature and hence is known to be carcinogenic. Replacement of chemical dyes with natural ones is the need of the hour in this era of global warming and environmental pollution. The aim of the study was to determine the efficacy of aqueous extract of turmeric and hibiscus as a cytoplasmic Aqueous stain of turmeric and hibiscus obtained from the rhizomes of Curcuma Longa and the petals of Hibiscus Rosa Sinesis respectively can be used as an alternate to eosin as an cytoplasmic stain.

Over the last decades, research on microbial bioerosion affecting archaeological bone assemblages highlighted the fact that this is a significant factor determining the long-term survival of vertebrate hard tissues as well as the quality of the biological information retrievable from them (e.g. isotopic, genetic, histomorphological). In spite of this, information about bioerosion is still scarce or inexistent for most regions around the world. Among the likely causes of this situation are the perceived and factual technical difficulties that surround the implementation of a standard research on this subject. Taking this into account, the aim of this paper is twofold: on the one hand, to describe a protocol for the preparation of bone samples (thick sections) suitable for observation with BSE-SEM that fulfils the criteria of simplicity, low cost and effectiveness; on the other hand, to present and discuss the first results derived from the application of such protocol to artiodactyl bone samples recovered at different Late Holocene archaeological sites from the southern Pampas of Argentina. The obtained results indicate that the implemented technique was effective in terms of providing good quality information at a very low cost as measured from the resources (time and materials) invested. In addition, the results show that a significant part of the analysed specimens exhibit extensive and intensive histological alteration compatible with the action of bacteria, which is unexpected in light of the currently prevailing model about the origin and conditions of the bacterial attack on animal bones in archaeological deposits (i.e. the so-called "endogenous model").

Background: Nature is vibrant and has an abundance of colors. The use of natural hues in histopathology could offer an economical and feasible alternative to the routinely used stains in special circumstances. Aim: To evaluate the efficacy of the extract of Hibiscus as a counterstain to hematoxylin. Methodology: Hibiscus calyces were sun dried and powdered. Alcoholic and water extracts were obtained by refluxing the powder with 95% ethanol and distilled water, respectively, for 3 h, and then cooled and filtered. Sections from 12 formalin-fixed paraffin-embedded tissue blocks, including both oral mucosal and skin tissues, were used. Nuclear staining was done with hematoxylin, following which the sections were stained with both alcoholic and aqueous Hibiscus extract for 10 min at room temperature using a dropper. All the slides were coded and were reviewed by three blinded oral pathologists for staining efficiency and intensity. NPar test, Kruskal-Wallis test, and Kappa statistics were done to assess the staining efficiency, intensity, and interobserver reliability for the selected parameters. Results: Statistically significant difference was not seen between Hibiscus extract and the controls, except in relation to staining of the basement membrane. Conclusion: Easily available and economical Hibiscus extract is an attractive alternative to eosin. Further studies involving the addition of mordants to the extract and its use as a special stain has to be explored.

Introduction: Oil immersion objective is not routinely used in histopathology and very rarely in cytopathology. We, on the other hand are routinely using the technique in our department on almost all types of lesions in histopathology and cytopathology. Objective: To determine the extra advantages of examining histopathology sections at 1000X magnification. Study Design: It was a comparative study. Settings: This study was carried out in department of pathology, Pakistan Institute of Medical Sciences, Islamabad. Duration of Study: June 2005 to Dec 2005. Material & Methods: Thirty diagnosed cases of gastric and colorectal adenocarcinomas were examined first under high power lens (400X) and then oil immersion lens using xylene (as an alternative for oil) for various parameters such as tumor architecture, cytoplasmic and nuclear features. The difference in various parameters was scored from 0-2. Results: The results revealed that nuclear features such as membrane breaks, nuclear pinchi...

This letter describes an innovative spin-coating system, built from off-the-shelf components, that can easily and inexpensively be integrated into any laboratory environment. Combined with a liquid suspension of conductive polymer, such a "rotary coater" enables simple coating of planar samples to create a physical protective barrier on the sample surface. This barrier aids in charge dissipation during scanning electron microscope and focused ion beam (FIB) imaging and provides wide-scale protection of the sample surface from ion bombardment during FIB imaging and gas-assisted deposition. This polymer layer replaces the localized and time-consuming electron beam deposition step typically performed during transmission electron microscopy lamella preparation. After observation, the coating can be easily removed, if desired. The described spin-coating procedure has minimal cost while providing repeatable positive results, without the need for expensive commercial coating inst...

Surface quality development on series of metal samples was investigated using a new Ar ion milling apparatus. The surface quality of samples was characterized by the image quality (IQ) parameter of the electron backscatter diffraction (EBSD) measurement. Ar ion polishing recipes have provided to prepare a surface appropriate for high quality EBSD mapping. The initial surfaces of samples were roughly grinded and polished. High quality surface smoothness could be achieved during the subsequent Ar ion polishing treatment. The optimal angles of Ar ion incidence and the polishing times were determined for several materials.

A simple cost effective, single stain, rapid method for the differential staining of structures of all the systems in histology including central nervous system applicable to paraffin sections is described. Several human tissues were fixed in 10% buffered neutral formalin. Paraffin sections were cut at 6 microns, slides were dewaxed, hydrated, and brought to water. Slides were stained for 3 minutes in the following staining solution. 0.25 grams of MV10B stain dissolved in 100 ml of autoclaved distilled water and filtered. Stained slides were washed in 95% alcohol, dehydrated, cleared and mounted in DPX. In central nervous system slides myelinated fibres and nerve cells were stained in varying shades of violet colour with a good colour value. In all the other systems slides the structures were well differentiated, gives an adequate contrast for observation and photography. As such this stain can be used as a broad spectrum stain in histology for all the systems. This single stain can...

We would like to report the case of a young lady with undiagnosed Wilson disease (WD) who presented with features of acute-on-chronic liver failure and died of its complications, which, if diagnosed in life, could have had a favorable evolution.

Surface quality development on series of metal samples was investigated using a new Ar ion milling apparatus. The surface quality of samples was characterized by the image quality (IQ) parameter of the electron backscatter diffraction (EBSD) measurement. Ar ion polishing recipes have provided to prepare a surface appropriate for high quality EBSD mapping. The initial surfaces of samples were roughly grinded and polished. High quality surface smoothness could be achieved during the subsequent Ar ion polishing treatment. The optimal angles of Ar ion incidence and the polishing times were determined for several materials.

While FIB sample preparation for transmission electron microscopy is a well established technique, few examples exist of samples of sufficient quality for atomic resolution imaging by aberration corrected (scanning) transmission electron microscopy (STEM). In this work we demonstrate the successful preparation of such samples from five different materials and present the refined lift-out preparation technique, which was applied here. Samples with parallel surfaces and a general thickness between 20 and 40 nm over a range of several mm were repeatedly prepared and analyzed by Cs-corrected STEM at 60 and 100 kV. Here, a novel 'wedge pre-milling' step helps to keep the protective surface layers intact during the whole milling process, allowing features close to or at the sample surface to be analyzed without preparation damage. Another example shows the cross-sectional preparation of a working thin film solar cell device to a final thickness of 10 to 20 nm over mm sized areas in the region of interest, enabling atomic resolution imaging and elemental mapping across general grain boundaries without projection artefacts. All sample preparation has been carried out in modern Dual-Beam FIB microscopes capable of low-kV Ga þ ion milling, but without additional preparation steps after the FIB lift-out procedure.

Lift-Out of TEM-Lamellae in most cases is done outside the FIB-System [1]. To investigate the advantages of recently introduced in-situ lift-out [2], we have integrated a compact and vacuum-compatible micromanipulator (Kleindiek Nanotechnik) into a LEO Gemini 1550 VP.

To demonstrate the cell wall of the given bacteria by CHANCE'S METHOD • THEORY: The cell wall of bacteria is a rigid structure which protects the cell against severe physical conditions and gives a definite shape to the cell. • The Gram positive bacterial cell wall consists of 20-80 nm thick peptidoglycan or murein layer with considerable amounts of teichoic acids and teichuronic acids as well as polysaccharides. • The Gram negative bacterial cell wall has a 1-3 nm peptidoglycan layer surrounded by a by a thick outer membrane containing lipoproteins and lipopolysaccharides.

We would like to report the case of a young lady with undiagnosed Wilson disease (WD) who presented with features of acute-on-chronic liver failure and died of its complications, which, if diagnosed in life, could have had a favorable evolution.

The long-term survival of many material types in the archaeological record is contingent on the objects' burial in favourable environments with which they can achieve some degree of equilibrium. This is especially true of organic artefacts. However, many burial environments evolve over time so that the sediments from which objects are eventually excavated may differ from those they experienced in the past. Many organic finds are so sensitive to changes in their chemical environment that their mere survival indicates that conditions have not changed dramatically since they entered the archaeological record. These materials are of limited use in understanding the history of an archaeological site's development. Skeletal materials, however, are composites comprising organic and inorganic phases in intimate association. Although subject to microbial and chemical degradation processes they differ from non-mineralized organics in that they persist in the archaeological record for thousands of years in a wide range of environmental conditions. These different degradation pathways leave very well-characterized changes in the mineralized tissues that can be used to identify different "diagenetic trajectories" and thus different environmental conditions. This work presents an SEM and chemical examination of bone, dentine and antler from three very different wet sites and compares these to both experimentally buried bones and skeletons with known burial histories. It is thus possible to reconstruct the burial histories of the finds from unknown environments.

Ancient bone represents an important resource for understanding early human societies and past economies. Human skeletal remains offer the most intimate link to our ancestors, providing important evidence on belief systems, burial rituals and social hierarchies, as well as contributing to our understanding of disease, diet and genetic affiliations in the distant past. Unfortunately, bone is prone to degradation by both biological and chemical agents. Bones from waterlogged environments undergo a variety of diagenetic changes depending upon the nature of the burial environment, and its condition on excavation can vary from almost perfectly preserved to a decalcified pseudomorph consisting of degraded and cross-linked collagen. As with other organic material from waterlogged environments, bone is prone to shrinkage and rapid deterioration if allowed to dry uncontrollably after excavation. Several workers have advocated their conservation using polyethylene glycols of various molecular weights, followed by air-or freeze-drying, a method that has seen considerable success in the conservation of bog bodies. For better preserved bone, impregnation with acetate emulsions (e.g. PVAC) or acrylate dispersions (PMMA/EA) are a common approach. Recent work suggests that prolonged washing, followed by 20% glycerol immersion and slow air drying is a viable alternative to traditional consolidation. The method is minimally invasive and may prove to not compromise future biomolecular or DNA analyses.