Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.

The ramus communicans, neural connection between medial and lateral plantar nerves of the horse, was transected to determine the degree to which medial and lateral plantar nerves contribute to the plantar ramus. After 2 months, sections of plantar nerves immediately proximal and distal to the communicating branch were collected and processed for electron microscopy. All examined nerves had undergone Wallerian degeneration and contained regenerating and mature fibers. Layers of the myelin sheath were separated by spaces and vacuoles, indicating demyelination of medial and lateral plantar nerves. Shrunken axons varied in diameter and were surrounded by an irregular axolemma. Shrunken axoplasm of both myelinated and non-myelinated fibers contained ruptured mitochondria and cristae, disintegrating cytoskeleton, and vacuoles of various sizes. The cytoplasm of neurolemmocytes contained various-sized vesicles, ruptured mitochondria within a fragile basal lamina and myelin whorls of multilayered structures indicative of Wallerian degeneration. These ultrastructural changes, found proximal and distal to the ramus in medial and lateral plantar nerves, suggest that axonal flow is bi-directional through the ramus communicans of the pelvic limbs of horses, a previously unreported finding. As well, maturity of nerves proximal and distal to the ramus indicates that all nerve fibers do not pass through the ramus.

Bryophytes are usually taken as good bioindicators. However, they represent a large group of terrestrial plants and they express an enormous range of peculiarities within the plant kingdom. With the aim to search for a common pattern of zinc binding, we established axenical in vitro cultures of a dozen bryophyte species that include hornworts, thallose, and leafy liverworts, as well as acrocarp and pleurocarp mosses. The species were grown free of contaminants for many years prior to the application of different treatments, i.e. offering Zn(II) from solid and liquid media and in combination with different anions. The localization and binding of zinc was detected by confocal microscopy using the zinc-specific dye FluoZin™-3. In one of the species, Hypnum cupressiforme (which is widely used for atmospheric heavy metal deposition studies in biomonitoring), semi-quantitative analyses of zinc were performed by energy dispersive X-ray microspectrometry (EDX) in a scanning electron microscope. The results suggest no common pattern of Zn(II) binding in different bryophyte species. Instead, the binding pattern seems to be species specific. Zinc is located in certain areas or cellular compartments, as clearly shown by the EDX measurements in H. cupressiforme.

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is an important vector for the xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which is associated with olive quick decline syndrome in southern Italy. The mouthparts of Hemiptera have important roles in host plant selection, feeding behavior and for vectoring pathogens that cause plant diseases. In this study, the functional morphology of the sensory structures located on the labium tip and precibarium of P. spumarius was investigated using scanning and transmission electron microscopy. The labium tip is composed of two symmetrical sensory complexes, each with five different types of sensilla: aporous sensilla trichodea type 1 and 2; uniporous sensilla chaetica type 1 and 2; and multiporous sensilla basiconica. The precibarium of P. spumarius has two kinds of sensory structures: bulbous sensilla and papillae sensilla. In particular, two groups of sensilla are located on the epipharynx: a distal group that consists of ten papillae sensilla and a proximal group composed of six papillae sensilla and two bulbous sensilla, while the hypopharynx has only two papillae sensilla. The involvement of these sensory structures in the context of feeding behavior and pathogen transmission is discussed.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.

The avian alimentary tract has evolved into different histologic structures to accommodate the physical and chemical features of several food types and flight requirements. We compared the esophagus, proventriculus, and gizzard of the domestic fowl, Gallus gallus domesticus (GGD) and kestrels, Falco tinnunculus (FT) using immunohistochemistry and scanning electron microscopy with various stains and lectins [Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin I (RCA120)], and α-smooth muscle actin (α-SMA). The esophagus of GGD demonstrated thickened epithelium, muscularis mucosae, and inner circular longitudinal tunica muscularis layers; moderate outer longitudinal tunica muscularis layers; and a true crop. In contrast, the esophagus of FT showed a thin epithelium, no muscularis mucosae, moderate inner longitudinal and thick outer circular tunica muscularis layers, and no true crop. In the proventriculus, the nature of the secretion in GGD was neutral, but that of FT was acidic and neutral. In the gizzard, the muscle coat of GGD by α-SMA had no muscularis mucosae, unlike FT, which had muscularis mucosae. In summary, there are many histologic differences between GGD and FT to meet their different physiologic needs, such as feeding.

Little is known of the olfactory mechanisms of host detection in the ovipositors of endoparasitoids and ectoparasitoids. An endoparasitoid Aprostocetus causalis La Salle & Wu (Hymenoptera: Eulophidae) and an ectoparasitoid Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) are the two parasitoids of the eucalyptus gall wasp Leptocybe spp. Structures and sense organs of ovipositors of A. causalis and Q. mendeli were studied using scanning and transmission electron microscopy, which provided essential information for exploring the mechanism of host detection by endoparasitoid and ectoparasitoid. The ovipositors of two parasitoids consisted of the first and second valvulae and ended in a pointed tip. There were three types of microtrichia, two types of sensilla chaetica, and one type of sensilla campaniformia on the ovipositors of A. causalis and Q. mendeli. However, Q. mendeli has the fourth type of microtrichia on the ovipositor. The morphology, types, distribution, length, and width of these sensilla and microtrichia were described, and their possible functions are discussed in conjunction with the stinging, oviposition, and the host selection process.

In the preparation of thin sections for microscopy, embedding and polishing processes in particular can change the composition and morphologies of samples. Soils and ashes are very fragile and solvent-susceptible, and appropriate sample preparation procedures have not been well-established. To improve the existing preparation methods and make them easier and faster, we embedded freeze-dried blocks, polished, and then examined these thin-section samples using polarization microscopy, laser microscopy, and field emission scanning electron microscopy with energy-dispersive X-ray spectrometry, and electron backscattered diffraction (EBSD). Appropriate thin-section samples can be prepared by: (1) rinsing with acetone and then embedding with Spurr resin along with repeated evacuation and ventilation, rather than conventional dehydration/replacement; (2) polishing using silicon carbide paper and diamond slurries, and then wiping with a cloth and a synthetic oil; and (3) slightly rinsing with 100% ethanol to remove the oil. The preparation method minimized contamination and pores, and showed flat surfaces and sometimes EBSD patterns. Freeze-drying has been claimed to cause the development of cracks due to ice crystal formation upon freezing, however, our method not only overcomes such problems for microscopic observation but saves substantial time, taking only 2 days in total to process a specimen, and requiring less than 1 g of resin and ~1 g of sample.

The present study planned to assess the medicinally important plants in the lower Margalla Hills of Islamabad, Pakistan using scanning electron microscopy of their pollens. Both quantitative and qualitative characters of the pollens were analyzed. The shapes of the pollen were prolate, spheroidal, elliptical, and oblate. Exine sculpturing showed rugulate, scabrate, psilate gemmate, and reticulate ornamentation. The people residing in the rural areas are thought to depend, primarily, on medicinal plant resources for many health care needs. A sample of 140 respondents was selected and interviewed by using a stratified random sampling technique. Results showed that the local communities of lower Margalla Hills are highly dependent on plants to treat various ailments, for example, respiratory (28%), digestive (46%) nervous, circulatory and dermatological problems (6% each), and reproductive problems (8%). The continuous dependence of the local community on ethnomedicinally important plants has led to serious degradation of the natural forest of lower Margalla Hills. Priority-based conservation and sustainable use of these forest resources are necessary, and use of alternative resources will also help in the forest conservation.

This work explores the combination of µ-Raman spectroscopy and scanning electron microscopy with X-ray energy dispersive spectrometry (SEM-EDS) for the study of the glazes in 15th–16th century Hispano-Moresque architectural tiles. These are high lead glazes that can be tin-opacified or transparent, and present five colors: tin-white, cobalt-blue, copper-green, iron-amber, and manganese-brown. They are generally homogenous and mineral inclusions are mostly concentrated in the glaze-ceramic interface. Through SEM-EDS, these inclusions were observed and chemically analyzed, whereas µ-Raman allowed their identification on a molecular level. K-feldspars, wollastonite and diopside were the most common compounds, as well as cassiterite agglomerates that render the glaze opaque. Malayaite was identified in green glazes, and andradite and magnesioferrite in amber glazes. Co–Ni–ferrites were identified in blue glazes, as well as Ni–Fe–olivines. Manganese-brown is the color where most compounds were identified: bustamite, jacobsite, hausmannite, braunite, and kentrolite. Through the µ-Raman analysis of different areas in large inclusions previously observed by SEM, it was possible to identify intermediate phases that illustrate the reaction process that occurs between the color-conferring compounds and the surrounding lead glaze. Furthermore, the obtained results allowed inference of the raw materials and firing temperatures used on the manufacture of these tiles.

This paper deals with the study of the surface morphology of barbels, upper lips, and adhesive discs in four hill stream fish species collected from Basistha River, a torrential river in Guwahati city, Assam, India. The four species belonging to the genus Garra namely, Garra gotyla (Gray, 1830), Garra gravelyi (Annandale, 1919), Garra stenorhynchus (Jerdon, 1849), and Garra nasuta (McClelland, 1838) were collected from the same torrential habitat. These fish revealed anatomical peculiarities in their barbels, lips, and adhesive discs which are believed to help them in adapting to such habitats. Organs exhibiting adaptive modifications (barbels, lips, and adhesive discs) were studied with the scanning electron microscope. The study primarily revealed the presence of type I and type II taste buds in the barbels, lips, and adhesive discs, and numerous unculi surrounded by microridges in the upper lips and adhesive discs. A cumulative, intercalated action of these organs enables these fish to adhere and adapt to rocky, torrential streams. Special ability to adapt to these habitats was reflected from the two types of taste buds (I and II) present in the barbels, and the clustering of excrescencies bearing unculi in the lips and adhesive discs of the fish.

Using the recently developed techniques of electron tomography, we have explored the first stages of disfiguring formation of zinc soaps in modern oil paintings. The formation of complexes of zinc ions with fatty acids in paint layers is a major threat to the stability and appearance of many late 19th and early 20th century oil paintings. Moreover, the occurrence of zinc soaps in oil paintings leading to defects is disturbingly common, but the chemical reactions and migration mechanisms leading to large zinc soap aggregates or zones remain poorly understood. State-of-the-art scanning (SEM) and transmission (TEM) electron microscopy techniques, primarily developed for biological specimens, have enabled us to visualize the earliest stages of crystalline zinc soap growth in a reconstructed zinc white (ZnO) oil paint sample. In situ sectioning techniques and sequential imaging within the SEM allowed three-dimensional tomographic reconstruction of sample morphology. Improvements in the detection and discrimination of backscattered electrons enabled us to identify local precipitation processes with small atomic number contrast. The SEM images were correlated to low-dose and high-sensitivity TEM images, with high-resolution tomography providing unprecedented insight into the structure of nucleating zinc soaps at the molecular level. The correlative approach applied here to study phase separation, and crystallization processes specific to a problem in art conservation creates possibilities for visualization of phase formation in a wide range of soft materials.

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.

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.

The antennal sensilla of female Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) were observed with scanning and transmission electron microscopy in this study. The antenna of Q. mendeli was geniculate, and the flagellum was composed of seven subsegments. Six distinct types of sensory receptors were observed, including sensilla basiconic capitate peg, sensilla böhm, sensilla chaetica, sensilla campaniformia, sensilla placodea and sensilla trichodea. Sensilla basiconic capitate pegs were found on the flagellomeres, and Böhm sensilla were found on the basal part of scape and the pedicel. Two morphological subtypes of sensilla chaetica were found on the antennae, and sensilla campaniformia were only found on the pedicel. Sensilla placodea were divided into two morphological subtypes that were found on the flagellomeres. Sensilla trichodea were found on the 2nd–6th flagellomere. By comparison to existing antennal sensilla, it was found that sensilla basiconic capitate peg, sensilla chaetica, sensilla placodea and sensilla trichodea were the most common sensilla of the parasitoids of Eulophidae. The external and internal morphology, types, number, distribution, length, and width of these sensilla were described, and their possible functions are discussed in conjunction with the host-detection behavior. Future studies on the host location mechanisms in Q. mendeli will be facilitated by these observations.

The distribution of lignin, 8-5′ and 8-8′ linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5′ linked lignin substructure), and no KM2 labeling (8-8′ linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5′ linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.