The desire to image specimens in liquids has led to the development of open-cell and closed-cell techniques in transmission electron microscopy (TEM). The closed-cell approach is currently more common in TEM and has yielded new insights into a number of biological and materials processes in liquid environments. The open-cell approach, which requires an environmental TEM (ETEM), is technically challenging but may be advantageous in certain circumstances due to fewer restrictions on specimen and detector geometry. Here, we demonstrate a novel approach to open-cell liquid TEM, in which we use salt particles to facilitate the in situ formation of droplets of aqueous solution that envelope specimen particles coloaded with the salt. This is achieved by controlling sample temperature between 1 and 10°C and introducing water vapor to the ETEM chamber above the critical pressure for the formation of liquid water on the salt particles. Our use of in situ hydration enables specimens to be loaded into a microscope in a dry state using standard 3 mm TEM grids, allowing specimens to be prepared using trivial sample preparation techniques. Our future aim will be to combine this technique with an in situ light source to study photocorrosion in aqueous environments.

Many studies have been carried out to investigate the morphological structure of the syrinx in many bird species. However, the cellular organization of the syrinx in the fowls and pigeons is still unclear. The current study revealed that in fowl and pigeon, the syrinx is formed of three main parts including tympanum (cranial) part, intermediate syringeal part, and bronchosyringeal (caudal) part, in addition to pessulus and tympaniform membranes. A great variation in the structural characteristics of syrinx of fowl and pigeon was recorded. In fowl, the tympaniform membranes showed a characteristic distribution of elastic and collagen fibers which increase the elasticity of tympaniform membranes. Moreover, the bony pessulus helps the medial tympaniform membranes to be stiffer, vibrate more strongly so that louder sound will be generated. In pigeon, the lateral tympaniform membrane is of greater thickness so that the oscillation of this membrane is reduced and the amplitude is lower. Moreover, the pessulus is smaller in size and is formed mainly of connective tissue core (devoid of cartilaginous or bony plates), resulting in the failure of stretching and vibrating of the medial tympaniform membranes, that leads to the generation of deeper sound. Electron microscopic examination of the syringes of fowls and pigeons revealed numerous immune cells including dendritic cells, plasma cells, mast cells, and lymphocytes distributed within syringeal mucosa and invading the syringeal epithelium. Telocytes were first recorded in the syrinx of fowls and pigeons in this study. They presented two long telopodes that made up frequent close contacts with other neighboring telocytes, immune cells, and blood capillaries.

We describe the morphological adaptations of the tongue and gastrointestinal tract of the striped sand snake Psammophis sibilans and discuss their functional importance. Using standard histological, histochemical, and scanning electron microscopy techniques, we analyzed 11 adult snakes of both sexes. Our findings showed that the bifurcated non-papillate tongue exhibited chemoreceptive adaptions to squamate foraging behavior. The lingual apex tapered terminally with sensory spines, and the body of the tongue possesses a characteristic central odor–receptor chamber that might serve to trap and retain scent molecules. Furthermore, the intrinsic musculature showed interwoven and well-developed transverse, vertical and longitudinal muscle fibers that control contraction and retraction during probing and flicking. The esophagus displayed highly folded mucosa lined with columnar epithelium with goblet cells. In contrast, the stomach mucosa formed finger-like gastric rugae, encompassing tubular glands with dorsal gastric pits. The intestine is distinct from other vertebrates in lacking the crypts of Lieberkühn in the tunica mucosa and submucosa. The intestine mucosa is mostly arranged in interdigitating villi oriented perpendicular to the luminal surface. We extrapolated subtle variations for both acid and neutral mucopolysaccharides and glycoproteins localization as well as collagen fibers using histochemical analyses. The elaborate histo-morphological and functional adaptation of the tongue and digestive tract plays a pivotal role in foraging and feeding behavior.

Post-testicular maturation of spermatozoa is crucial for attaining the morphological and functional capabilities needed for successful fertilization. Epididymal epithelia offer a favorable environment for spermatozoa that are stored long term in the turtle epididymis; however, sperm–epithelial interactions during storage, which are enormously important for sperm functional and morphological maturation, are still largely unknown in turtles. The present study examined the epididymis during the sperm-storage period (November–April) in the Chinese soft-shelled turtle (Pelodiscus sinensis). Light and transmission electron microscopy were used to determine the cellular features of each epididymal segment (caput, corpus, and cauda) and their epithelial interactions with the spermatozoa. Spermatozoa were mainly located in the lumena of caput, corpus, and cauda epididymides. Numerous spermatozoa were bound to apical surfaces of the epithelia, and several were even embedded in the epithelial cytoplasm of the caput and corpus epididymides. No embedded spermatozoa were found in the cauda epididymis. In all epididymal segments, principal and clear cells showed the synthetic activity, evidenced by a well-developed endoplasmic reticulum network and high and low electron-dense secretory materials, respectively. Principal and clear cells in the caput and corpus segments showed embedded spermatozoa in electron-dense secretions and in the lipid droplets within the cytoplasm. No lysosomes were observed around the embedded spermatozoa. The lumena of the caput and corpus segments showed few apocrine and low electron density secretions. In the lumen of the cauda epididymidis, different secretions, such as holocrine with low and high electron density and their fragmentation, apocrine, and dictyosome, were found and are summarized. Altogether, sperm physical interactions with secretions either in the cytoplasm of epithelium or in the lumen may support the viability, morphological maintenance, and transfer of various proteins involved in long-term sperm storage in the turtle. This interaction could help us to understand the mechanisms of long-term sperm storage and provide more insights into the reproductive strategies of turtle sperm preservation.

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

The carpenter ant Camponotus rufipes has intracellular bacteria in bacteriocytes scattered in the midgut epithelium, which have different amounts of endosymbionts, according to the developmental stages. However, there are no detailed data about the midgut cells in adult workers. The present work aimed to evaluate the morphology and cellular events that coordinate the abundance of endosymbionts in the midgut cells in C. rufipes workers. The midgut epithelium has digestive cells, bacteriocytes, and cells with intermediate morphology. The latter is similar to bacteriocytes, due to the abundance of endosymbionts, and similar to digestive cells, due to their microvilli. The digestive and intermediate cells are rich in autophagosomes and autolysosomes, both with bacteria debris in the lumen. These findings suggest that midgut cells of C. rufipes control the endosymbiont level by the autophagy pathway.

Hymenolepis nana, typically a parasite found in conventionally established mouse colonies, has zoonotic potential characterized by autoinfection and direct life cycle. The objective of this study was to determine the rate of parasite infection in laboratory mice. The hymenolepidide cestode infected 40% of the 50 mice sampled. The rate of infection in males (52%) was higher than in females (28%). Morphological studies on the cestode parasite showed that worms had a globular scolex with four suckers, a retractable rostellum with 20–30 hooks, and a short unsegmented neck. In addition, the remaining strobila consisted of immature, mature, and gravid proglottids, irregularly alternating genital pores, lobulated ovaries, postovarian vitelline glands, and uteri with up to 200 eggs in their gravid proglottids. The parasite taxonomy was confirmed by using molecular characterization based on the sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (mtCOX1) gene. The parasite recovered was up to 80% identical to other species in GenBank. High blast scores and low divergence were noted between the isolated parasite and previously described H. nana (gb| AP017666.1). The phylogenetic analysis using the COX1 sequence places this hymenolepidid species of the order Cyclophyllidea.

Studying the dermal skeleton in fish is valuable for phylogenetic specification. The current study describes the detailed structure of the plecostomus dermal skeleton, including its morphogenesis and distribution in the skin. The denticles have a crown and a basal part and are embedded in bony depressions, to which they are attached by denticle ligaments. During denticle morphogenesis, denticle papillae formed from denticle precursor cells align in two cellular layers: an outer ameloblast precursor layer and an inner odontoblast precursor layer. The ameloblast precursors and odontoblast precursors differentiate and secrete enamel and dentine, respectively. We used different histochemical techniques, including Crossmon's trichrome staining, Weigert–Van Gieson staining, periodic acid–Schiff (PAS) staining, combined Alcian blue (AB; pH 2.5)/PAS staining, Weigert–Van Gieson staining, Mallory trichrome staining, and AB staining to distinguish the dentine and denticle ligaments. We used acridine orange to detect lysosome activity during denticle eruption. Transmission electron microscopy was used to detect the denticle ultrastructure, and scanning electron microscopy was used to detect the topographic distributions of different types of dermal tissues in different anatomical regions.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.