Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples

Specimen preparation methods based on high‐pressure freezing and freeze‐substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy. However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast. By contrast, some efforts to increase membrane staining may lead to excessively granular staining. No one freeze‐substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis. However, one or more of the following protocols, perhaps with minor modifica‐tions, should yield satisfactory results in most cases. Freeze‐substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high‐resolution structural studies. Membranes are typically lightly stained but very well defined. Initial freeze‐substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes. Finally, initial trials indicate that freeze‐substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes. The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze‐substitution reagent.     

Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome,Picrosirius red and confocal microscopy techniques

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin‐Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image‐Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E‐confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland‐Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E‐confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E‐confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.     

Structural study of the salivary glands of Anocentor nitens (Acari: Ixodidae) during the feeding cycle

The salivary glands of Anocentor nitens (Neumann, 1897 ) occur in pairs and are located in the anterolateral region of the general cavity, with milky white color and approximately equal sizes. They consist of a secretory portion and an excretion duct. In some glandular acini, all the cells had a basophilic appearance they were stained by hematoxylin, whereas others presented cells with different staining affinities. In this work, we describe the variations observed in these glands during the feeding cycle of ticks [after feeding (0 h) and successively at 24, 48, 72, 96, 120, and 144 h]. The cells stained by hematoxylin were shown to be more reactive to Alcian blue, thus demonstrating the presence of acid glycosaminoglycans, whereas those stained using eosin presented weak or no reaction. A strong reaction was found by the use of the periodic acid‐Schiff (PAS) technique, thereby suggesting the presence of glycogen and/or glycoconjugates containing hexose, confirmed by using salivary amylase before PAS, with partial destaining of the slides. Continuing presence of residual staining in these cells suggests the presence of glycoconjugates containing hexose. Cells with nuclei of circular outline and few granules (of different sizes) were found in type II acini, 72 h after collection. Type I acini presented wide lumina and walls composed of larger numbers of cells of cubic to cylindrical shape. The pronounced degranulation shown in this study over the course of the feeding cycle was associated with the release of substances for oviposition. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Morphological and microscopic identification of three major medicinal Dendrobium species in Ta‐pieh Mountains area

Dendrobium is an important medicinal material in China. It has the effect of nourishing the stomach, nourishing yin and clearing heat. In China, there are many types of Dendrobium, and different Dendrobium species have different efficacy. The present study is aimed at distinguishing three major Dendrobium species from morphological and microscopic identification in Ta‐pieh mountains area. In this article, the roots, stems and leaves of Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme are used as materials to compare the differences of tissues of these three Dendrobium species by morphological indexes and microscopic identification of different Dendrobium. The stem morphology of these three Dendrobium species was significantly different except for stem internodes number and the middle part of the stem diameter by measuring the stems of Dendrobium. To ensure the safe use of Dendrobium, we built a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. huoshanense, D. officinale, and D. moniliforme. The microscopic results show that different types of Dendrobium exhibit different states that can be distinguished under normal light normal and fluorescence microscopy. This comparative study of morphology and microscopy contributes to the development of identification and quality evaluation of Dendrobium.     

Microscopic and molecular evidence in support of rodent as a reservoir for dissemination of Leishmaniasis

Leishmaniasis is a worldwide public health problem and vector‐borne disease. It is caused by a diverse group of protozoan parasites that belong to the genus Leishmania and transmitted to humans through a bite of an infected female sand fly. Leishmaniasis has attained epidemic proportion in Khyber Pakhtunkhwa and raises serious concern over its management. The present research work was conducted in cutaneous leishmaniasis (CL) prevalent village named Surgul of district Kohat, Khyber Pakhtunkhwa with a focus to investigate whether rodent can act as a source for dissemination of leishmanial species or not. In this context, rodent samples were analyzed via morphological and molecular approaches to unveil prevalence of CL. It was reported that 12.5% of samples were positive for signs of leishmaniasis through microscopy and 18.75% through polymerase chain reaction (PCR). Supporting the findings further, the color character of rodents was also taken into consideration, which shows that light dark colored rodents were more infected (13.3%) compared to brown colored rodents (11.43%). Based on our findings, we speculate that small rodents are a possible reservoir of various leishmanial parasites and play a significant role in zoonosis and maintenance of their species.     

Plasmodium species aware based quantification of malaria parasitemia in light microscopy thin blood smear

Malaria is a serious worldwide disease, caused by a bite of a female Anopheles mosquito. The parasite transferred into complex life round in which it is grown and reproduces into the human body. The detection and recognition of Plasmodium species are possible and efficient through a process called staining (Giemsa). The staining process slightly colorizes the red blood cells (RBCs) but highlights Plasmodium parasites, white blood cells and artifacts. Giemsa stains nuclei, chromatin in blue tone and RBCs in pink color. It has been reported in numerous studies that manual microscopy is not a trustworthy screening technique when performed by nonexperts. Malaria parasites host in RBCs when it enters the bloodstream. This paper presents segmentation of Plasmodium parasite from the thin blood smear points on region growing and dynamic convolution based filtering algorithm. After segmentation, malaria parasite classified into four Plasmodium species: Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, and Plasmodium malaria. The random forest and K‐nearest neighbor are used for classification base on local binary pattern and hue saturation value features. The sensitivity for malaria parasitemia (MP) is 96.75% on training and testing of the proposed approach while specificity is 94.59%. Beside these, the comparisons of the two features are added to the proposed work for classification having sensitivity is 83.60% while having specificity is 94.90% through random forest classifier based on local binary pattern feature.     

Microwave-enhanced fixation of rabbit articular cartilage

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave-enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 × 7 × 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo- or microwave-enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave-enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave-enhanced fixation is equal in quality to the best conventional immersion fixation and is nearly as fast as cryo-preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.     

Imaging of extraradicular biofilm using combined scanning electron microscopy and stereomicroscopy

Microorganisms are able to survive and induce persistent infection in extraradicular areas. The objective of this study was to evaluate the relationship between extraradicular biofilm and persistent periapical periodontitis. Thirty‐five apical samples with different stages of pulp and periapical pathology (vital pulp, pulp necrosis without radiographically visible periapical lesions, chronic periapical periodontitis that had not received root canal therapy and persistent periapical periodontitis) were initially evaluated using scanning electron microscopy. The same samples were then processed using Brown and Brenn‐modified Gram staining. We detected extraradicular biofilm in all samples with persistent periapical periodontitis and in three samples with chronic periapical periodontitis. The extraradicular bacteria predominantly had rod and filament morphology and were surrounded by varying amounts of amorphous extracellular material. The surfaces outside the root of the apical samples with vital pulp and pulp necrosis were covered by fibers, and no extraradicular microorganisms were present, which suggests that extraradicular biofilm is closely associated with failed endodontic treatments, thus resulting in persistent infection. Microsc. Res. Tech. 76:979–983, 2013. © 2013 Wiley Periodicals, Inc.     

Morphological and chemical changes in human deciduous dentin after phosphoric acid,self‐etching adhesive and Er: YAG laser conditioning

The morphological and chemical changes in deciduous dentin produced by different conditioning protocols were evaluated in this in vitro study. Eighty primary dentin samples were divided into eight groups (n = 10): G1, acid etching; G2, self‐etching adhesive; G3, G4, Er: YAG laser irradiation at 25.5 and 38.2 J cm^?2, respectively; 10 Hz and spray irrigation. Groups 5, 6, 7, and 8 were irradiated at previous densities, and then phosphoric acid or self‐etching adhesive conditioning was applied. Scanning electron microscopy (SEM) and energy dispersive X‐ray spectroscopy (EDS) were used to evaluate chemical and morphological changes. Paired t‐test and One‐way ANOVA were used for statistical analysis (p ≤ 0.05). All samples showed different morphology with specific characteristics according to the conditioning protocol. Changing element concentration values are expressed in atomic percent (at %). After conditioning, there were statistically significant differences (p ≤ 0.05) for p at% and Ca/P in all groups; highlighting the following additional findings by group: G1, G7, and G8 showed changes in all elements studied, G2 presented a decrease in C at% and increased Ca at%, G3 and G4 exhibited at% changes in C, trace elements and Ca. Furthermore, G5 showed at% changes in O and trace elements; while G6 changes were observed on C at%, O at% and trace elements at%. Dentin morphology and chemical composition varied in accordance with the conditioning protocol, with characteristics specific for each one that could have clinical implications for the retention and bond strength performance of adhesive materials.     

Foliar epidermal anatomical characteristics of taxa of Iris subg. Scorpiris Spach (Iridaceae) from Turkey

Iris L. is one of the important genus of family Iridaceae, consist of 56 taxa naturally occurred in Turkey. The similarities and variations in the subgenus overlapping the taxonomic positions of the species in the subgenera and needs anatomical assessment especially by microscopic techniques. In this study, the taxonomic significance of leaf anatomical characters of 10 Iris subgenus Scorpiris taxa were studied in detail and the relationship among these taxa were evaluated using microscopy techniques. Fresh leaf samples of species were fixed in 70% alcohol solution for anatomical observation under microscope. Eleven different micromorphological features were statistically analyzed to delimit the species in subgenus. Based on morphological and anatomical similarities, we studied relationships among; (1) ssp. turcica, ssp. caucasica, I. nezahatiae and I. pseudocaucasica; (2) correlation between ssp. turcica and ssp. caucasica; (3) association of I. galatica, I. persica, ssp. margaretiae and ssp. stenophylla with each other; (4) relationship between ssp. stenophylla and ssp. margaretiae; and (5) relevance between I. aucheri and I. peshmeniana. Moreover, the taxonomy of subgenus Scorpiris has been discussed in detail with novel and diagnostic features based on micromorphological physiognomies. We found that four species in this study are endemic to Turkey, while seven are critically endangered geophytes in the country. The leaf anatomical characteristics of 10 taxa were divided into three groups. Main aim of this research was to study the taxonomy of the complex subgenus Scorpiris through microscopic techniques.     

Identification of Hypoderma actaeon (Diptera: Oestridae) in red deer (Cervus elaphus) from northern Spain: Microscopy study and molecular analysis

Hypoderma spp. larvae were observed subcutaneously in the dorsal and lumbar regions of red deer (Cervus elaphus) hunted in the province of León (northwestern Spain) causing a myiasis. They were removed and initially classified by their size, shape, color, and location under the skin into the three larval stages that parasitize these animals. The morphological characteristics of the first and second-instar are described and from the features of the third-instar the species was identified as Hypoderma actaeon. To accurately identify this species, five isolates of genomic DNA from the third-instar, two from the second-instar and two from first-instar of H. actaeon were analyzed by PCR analysis of the COI region of mt-DNA. The results confirmed that the examined samples exactly matched with H. actaeon. This study has shown the morphological identification of the three larval stages of H. actaeon and, for the first time, the first and second-instar larvae have been molecularly characterized. Finally, identification of only H. actaeon suggests that this species is the only affecting red deer in the Iberian Peninsula.     

Rapid and reliable method for diagnostic electron microscopy of faeces

Four methods are described for examining viruses in faeces by electron microscopy using negative staining. Faeces samples from a total of 180 patients with diarrhoea illnesses were processed for electron microscopy by the direct staining technique, pseudoreplica technique, microsolute concentration technique and by ultracentrifugation. Virus particles (including rotaviruses and adenoviruses) were found in fifty-five (31%) samples when the results of all methods were combined. Bacteriophages were found in thirty of the samples which were read virus negative. Herpesviruses were also found in the faecal samples of two patients with diarrhoea illness. Parvovirus-like particles were identified in one sample which was rotavirus and adeno-virus positive. Calicivirus-like particles were found in a sample which was adenovirus positive. With the direct staining technique thirty-six (20%) samples contain virus particles; with micro-solute concentration technique forty-eight (27%) samples contain virus particles and with ultracentrifugation thirty-eight (21%) contain virus particles. It is believed the microsolute concentration technique is rapid and more reliable than the pseudoreplica technique and ultracentrifugation method, and is a more preferable method for the diagnosis of faecal sample by electron microscopy.     

Ultrastructural changes in the cemento‐enamel junction caused by acidic beverages: An in vitro study

The present in vitro study was aimed at evaluating the morphological changes in the cemento‐enamel junction (CEJ) after exposure to acidic beverages using the scanning electron microscopy (SEM). The initial pH and titratable acidity (TA) was analyzed from follow groups: (I) Coca cola, (II) orange juice, (III) Cedevita, (IV) Red Bull, (V) Somersby cider, and (VI) white wine. The CEJ samples (n = 64), obtained from unerupted third molars, were allocated to one control (artificial saliva, n = 16) and six experimental groups (n = 8). The experimental samples were immersed in beverages (50 ml) for 15 min, three times daily, 10 days, and in artificial saliva between immersions. SEM analysis was performed in a blind manner, according to scoring scale. One‐way ANOVA and Tukey's post hoc tests, as well as Kruskal–Wallis and Mann–Whitney U test used for statistical analysis. The pH values of the acidic beverages ranged from 2.65 (Coca cola) to 3.73 (orange juice), and TA ranged from 1.90 ml (Coca cola) to 5.70 ml (orange juice) of NaOH to reach pH 7.0. The SEM analysis indicated statistically significant differences between the control samples and those immersed in acidic beverages. The Groups IV, I, and II, showed the highest CEJ damage grade while those of the Group VI were the lowest. All the tested acidic beverages caused morphological changes in the CEJ with a smaller or larger exposure of dentine surface, and were not always related to the pH or TA of acidic beverages.     

The effect of gastric acid on chitosan modified glass ionomer cement: SEM‐EDS

This study aimed to use scanning electron microscopy with energy dispersive spectroscopy (SEM‐EDS) to examine the elements that passed into the gastric acid solution after the application of a gastric acid erosive cycle to chitosan modified glass ionomer cement (GIC). Chitosan modified GIC samples were obtained by adding chitosan (vol/vol) of 5 and 10% to GIC for the experimental groups. These two experimental groups and a control group were subjected to gastric acid erosive treatment for 60 s six times a day for 10 days. The sample surfaces were coated with approximately 1 nm of gold to increase conductivity with the Q 150R ES device (Quorum Technologies, East Sussex, England). Surface topography images were obtained with a SEM. Besides, EDS analysis was also determined the number of elements graphically in the region where the fast electron beam hit. In the samples examined, the amount of element was determined. After gastric acid application, cracks and voids were observed on the surfaces of the samples. In the EDS analysis of the 5 and 10% chitosan modified GIC and control groups, Si, Al, Na, and F was found. It is necessary to investigate the antibacterial properties and physical properties of chitosan modified glass ionomer‐free elements and fluorine ions using advanced techniques.     

Morphological changes produced by acid dissolution in Er:YAG laser irradiated dental enamel

Several scientific reports have shown the effects of Er:YAG laser irradiation on enamel morphology. However, there is lack of information regarding the morphological alterations produced by the acid attack on the irradiated surfaces. The aim of this study was to evaluate the morphological changes produced by acid dissolution in Er:YAG laser irradiated dental enamel. Forty‐eight enamel samples were divided into four groups (n = 12). GI (control); Groups II, III, and IV were irradiated with Er:YAG at 100 mJ (12.7 J/cm²), 200 mJ (25.5 J/cm²), and 300 mJ (38.2 J/cm²), respectively, at 10 Hz without water irrigation. Enamel morphology was evaluated before‐irradiation, after‐irradiation, and after‐acid dissolution, by scanning electron microscopy (SEM). Sample coating was avoided and SEM analysis was performed in a low‐vacuum mode. To facilitate the location of the assessment area, a reference point was marked. Morphological changes produced by acid dissolution of irradiated enamel were observed, specifically on laser‐induced undesired effects. These morphological changes were from mild to severe, depending on the presence of after‐irradiation undesired effects. Microsc. Res. Tech. 77:410–414, 2014. © 2014 Wiley Periodicals, Inc.     

A new technique for perfusion fixation and contrast enhancement of foetal lamb myocardium for electron microscopy

A technique of perfusion fixation following elective diastolic cardiac arrest was developed to obtain excellent preservation of foetal lamb hearts for morphological studies with electron microscopy. A technique was developed to markedly enhance the contrast of embedded material by treating thin sections with tannic acid prior to lead staining. This technique has been particularly useful for quantitative morphometric analysis.     

Effect of <i>Lycopus lucidus</i> Turcz. supplementation on gut microflora and short chain fatty acid composition in Crj: CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice. In addition, we evaluated the production of major cytokines (Interleukin-6 and -10) which are related to inflammation and fatty acid composition of several tissues. 16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base. Male mice fed on L. lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group (p < 0.05). 16S rDNA sequencing analysis of fecal samples showed that L. lucidus supplementation decreased the community of harmful microflora (Enterobacteriaceae including Escherichia coli and Bacteroides sp.) in feces compared with the control group (p < 0.05). There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L. lucidus fed groups. The fecal fatty acid composition in the L. lucidus group had percentages of 4:0, 6:0, 8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group. Our results showed that L. lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.     

Potential of a sensitive uric acid biosensor fabricated using hydroxyapatite nanowire/reduced graphene oxide/gold nanoparticle

In this study, a ternary nanocomposite consisting of gold nanoparticles (AuNPs), hydroxyapatite (HAP) nanowires, and reduced graphene oxide (rGO) is synthesized by a simple one‐step hydrothermal method, which is used to modify glassy carbon electrode (GCE) for detecting uric acid. The nanocomposite is characterized through various methods such as scanning electron microscopy, transmission electron microscopy, and X‐ray diffraction. Electrochemical measurements of the modified GCE are performed in a conventional three‐electrode system. Experimental results show that the obtained HAP nanowire and rGO are mixed homogeneously, and the AuNPs are deposited into this matrix. The GCE modified by the nanocomposites have superior electrocatalytic activities for uric acid. The peak current intensities of UAO (uricase)/HAP‐rGO/AuNPs sensing system linearly increase as the uric acid concentration increases substantially in a range of 1.95 × 10^?5 to 6.0 × 10^?3 M (R² = .9943), with a detection limit of 3.9 × 10^?6 M (S/N = 3) and analytical sensitivity of 13.86 mA/M. The biosensor performs well in determining uric acid concentration in human urine samples.