Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500μm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.

The Loranthaceae Juss. family includes parasitic species that invade important trees such as fruit trees. In Saudi Arabia, Loranthaceae comprises four genera, which include six species that grow in the western, southwestern, and northern regions: Tapinanthus globifer (A.Rich.) Tiegh, Oncocalyx glabratus (Engl.) M. G. Gilbert, Loranthella deflersii (Tiegh.) S. Blanco & C. E. Wetze, Phragmanthera austroarabica A. G. Mill. & J. Nyberg, Plicosepalus curviflorus (Benth.ex Oliv.) Tiegh. and Plicosepalus acaciae (Zucc.). The species present in the Kingdom of Saudi Arabia have not been the subject of enough studies. This work aims to screen and evaluate the taxonomic importance of the micromorphological traits of leaves and fruits in Loranthaceae species native to Saudi Arabia (SA) using scanning electron microscope (SEM). In this study, cluster dendrogram (CD), principal component analysis (PCA) and analysis of variance (ANOVA) were used to evaluate the ability to discriminate Loranthaceae species using micromorphological characteristics. Most of the micromorphological characteristics of the leaf and fruit surfaces used reflected significant variation between the species of Loranthaceae. The type of stomata, trichome, lenticels, fine relief of the cell wall and wax form were the most taxonomically important characteristics. In addition, the cluster dendrogram of morphological characteristics showed species distribution within branches based on affiliation to subtribes Tapinanthinae and Emelianthinae. To the best of our knowledge, the fruit and leaves of the species under study have never been described using electron microscopy, and this study is considered the first of its kind. It also contributes to solving the classification problems of the family Loranthaceae in general and confirms the importance of the characteristics and methods used as tools for characterizing parasitic species that infect trees and helps to verify their identities. This will help to improve resistance efforts and put effective control plans in place.

The retina consists of various cell types arranged in eight cell layers and two membranes that originate from the neuroectodermal cells. In this study, the timing of differentiation and distribution of the cellular components and the layers of the rabbit retina are investigated using light and electron microscopy and immunohistochemical techniques. There were 32 rabbit embryos and 12 rabbits used. The rabbit retina begins its prenatal development on the 10th day of gestation in the form of optic cup. The process of neuro- and gliogenesis occurs in several stages: In the first stage, the ganglionic cells are differentiated at the 15th day. The second stage includes the differentiation of Muller, amacrine, and cone cells on the 23rd day. The differentiation of bipolar, horizontal, and rod cells and formation of the inner segments of the photoreceptors consider the late stage that occurs by the 27th and 30th day of gestation. On the first week of age postnatally, the outer segments of the photoreceptors are developed. S100 protein is expressed by the Muller cells and its processes that traverse the retina from the outer to the inner limiting membranes. Calretinin is intensely labeled within the amacrine and displaced amacrine cells. Ganglionic cells exhibited moderate immunoreactivity for calretinin confined to their cytoplasm and dendrites. In conclusion, all stages of neuro- and gliogenesis of the rabbit retina occur during the embryonic period. Then, the retina continues its development postnatally by formation of the photoreceptor outer segments and all layers of the retina become established. RESEARCH HIGHLIGHTS: The aim of this study is to investigate the morphogenesis of the rabbit retina during pre- and postnatal life. The primordia of the retina could be observed in the form of the optic cup. The ganglionic cells are the first cells to differentiate, while the photoreceptor cells are the last. S100 protein is expressed by the Muller cells and its processes. Calretinin is intensely labeled in the amacrine and displaced amacrine cells and moderately expressed in the cytoplasm and dendrites of ganglionic cells.

Scales are structures composed of mineralized collagen fibrils embedded in the skin of fish. Here we investigate structures contributing to the bulk of the scale material of the sturgeon (Acipencer guldenstatii) at the millimeter, micrometer and nanometer length scales. Polished and fracture surfaces were prepared in each of the three anatomic planes for imaging with light and electron microscopy, as well as focused ion beam - scanning electron microscopy (FIB-SEM). The scale is composed of three layers, upper and lower layers forming the bulk of the scale, as well as a thin surface layer. FTIR shows that the scale is composed mainly of collagen and carbonated hydroxyapatite. Lacunae are present throughout the structure. Fracture surfaces of all three layers are characterized by large diameter collagen fibril bundles (CFBs) emanating from a plane comprising smaller diameter CFBs orientated in different directions. Fine lineations seen in polished surfaces of both major layers are used to define planes called here the striation planes. FIB-SEM image stacks of the upper and lower layers acquired in planes aligned with the striation planes, show that CFBs are oriented in various directions within the striation plane, with larger CFBs emanating from the striation plane. Fibril bundles oriented in different directions in the same plane is reminiscent of a similar organization in orthodentin. The large collagen fibril bundles emanating out of this plane are analogous to von Korff fibrils found in developing dentin with respect to size and orientation. Scales of the sturgeon are unusual in that their mineralized collagen fibril organization contains structural elements of both dentin and bone. The sturgeon scale may be an example of an early evolved mineralized material which is neither bone nor dentin but contains characteristics of both materials, however, the fossil data required to confirm this is missing.

The 20th century unfolded many mysteries regarding renal pathology with the advent of ancillary techniques such as immunofluorescence (IF) and electron microscopy (EM) studies. EM plays a major role in confirming the routine and IF findings or may uncover new and unsuspected features. The aim of the study is to elucidate the role of ultrastructural findings in patients with medical kidney diseases on whom kidney biopsy was performed at the American University of Beirut Medical Center, between November 2018 and June 2019. A total of 188 renal biopsies were examined during the study period. EM confirmed the light microscopy diagnosis in 54% of the cases while completely changed the diagnosis in 23% of the cases. In 23% of the sample, EM provided additional features and a secondary diagnosis. Our study emphasizes the important diagnostic role of EM and its significance, particularly in minimal change disease, basement membrane abnormalities, and glomerulopathies.

This study elucidated the etiology of C3 glomerulonephritis (C3GN) and non-C3GN with primary membranoproliferative glomerulonephritis (MPGN) using transmission electron microscopy (TEM) and periodic acid-methenamine silver stain (PAM-EM). Thirty-one primary MPGN cases were analyzed by TEM and PAM-EM to distinguish among MPGN I, MPGN II, MPGN III Burkholder subtype (MPGN IIIB), and Anders and Strife subtype (MPGN IIIA/S). Each case was also classified into C3GN or non-C3GN according to the standard C3GN definition using immunostaining. Four cases of MPGN II met C3 glomerulopathy; whereas, four cases of MPGN IIIB did not meet C3 glomerulopathy. Seven of 11 cases (64%) of MPGN I without GBM disruption and 7 of 12 cases (58%) of MPGN IIIA/S with GBM disruption met the non-C3GN criteria with significant immunoglobulins\' deposition. Regardless of the C3GN or non-C3GN diagnosis, the deposits in primary MPGN I and MPGN IIIA/S exhibited ill-defined, amorphous, and foggy characteristics similar to those found in postinfectious GN but were different from immune complex (IC) deposits seen in MPGN IIIB. Not only C3GN but also non-C3GN was due to mechanisms other than IC deposition as found in postinfectious GN. Consequently, GBM disruption of MPGN IIIA/S was not due to IC deposition.

UNASSIGNED: This study aimed to evaluate enamel surface roughness and microhardness following the use of different bracket materials (metal or ceramic), etchants (total- and self-etchants), and adhesive systems (precoated or flash-free).
UNASSIGNED: A total of 99 extracted human premolars were selected for the analysis. The surface roughness was first assessed (roughness control). One specimen from each subgroup was examined using a scanning electron microscope to illustrate the surface topography. Eighty-eight teeth were prepared using total- or self-etchants and bonded to precoated or flash-free adhesive metal or ceramic brackets. The remaining 11 specimens were not bonded to brackets (microhardness controls). The brackets were debonded after immersion in distilled water for 24 h. The specimens were again scanned for surface roughness and topography imaging. Finally, the microhardness was assessed using a micro-Vickers hardness test at a force of 200 g for 10 s.
UNASSIGNED: An overall statistically significant increase in surface roughness and reduced surface microhardness were observed in all experimental groups when compared with those in the control groups. The etchant type was the only variable found to contribute to the measured surface properties, with increased roughness and reduced microhardness introduced by total-etching compared to those by self-etching.
UNASSIGNED: Orthodontic brackets introduced a significant increase in enamel surface roughness and reduce microhardness compared with untreated enamel, regardless of the bracket material, etchant type, and adhesive system. The etchant type was the only variable contributing to these changes, with total etching having a more pronounced effect.

BACKGROUND: Histology of human oral mucosa is closely related with its function and anatomical location, and a proper characterization of the human masticatory oral mucosa could be very useful in periodontal pathology.
OBJECTIVE: In the present work, we have carried out a comprehensive study in order to determine the main histological features of parakeratinized (POM) and orthokeratinized (OOM) masticatory human oral mucosa using light and electron microscopy.
METHODS: To perform this, we have used several histological, histochemical and immunohistochemical methods to detect key markets at the epithelial, basement membrane and connective tissue levels.
RESULTS: Our results demonstrated that POM and OOM share many histological similarities, as expected. However, important differences were observed at the epithelial layer of POM, that was significantly thicker than the epithelial layer found in OOM, especially due to a higher number of cells at the stratum spinosum. The expression pattern of CK10 and filaggrin revealed intense signal expression in OOM as compared to POM. Collagen and proteoglycans were more abundant in OOM stroma than in POM. No differences were found for blood vessels and basement membrane.
CONCLUSIONS: These results may contribute to a better understanding of the pathological conditions affecting the human masticatory oral mucosa. In addition, these findings could be useful for the generation of different types of oral mucosa by tissue engineering techniques.
UNASSIGNED: Microscopical features of parakeratinized and orthokeratinized masticatory human oral mucosa showed important differences at both, epithelial and stromal levels. Parakeratinized masticatory human oral mucosa exert thicker epithelial layer, especially, at the stratum spinosum in comparison to orthokeratinized human oral mucosa. Cytokeratin 10 and filaggrin human epithelial markers were intensively expressed in orthokeratinized masticatory human oral mucosa in comparison to parakeratinized masticatory human oral mucosa. At the stromal level, orthokeratinized masticatory human oral mucosa exhibit higher levels of collagen and proteoglycans than parakeratinized masticatory oral mucosa. The deep knowledge of histological features of masticatory oral mucosa could lead to a better understanding of oral mucosa pathology and advanced treatments.

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria-vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes.