Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate: light and transmission electron microscopical observations.

OBJECTIVES The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two commercially available calcium phosphate bone substitute materials retrieved from bone defects. MATERIAL AND METHODS Six defects were prepared bilaterally in the mandibular body of three mini pigs. The defects were randomly grafted with either deproteinized bovine bone mineral (DBBM) or biphasic calcium phosphate (BCP). After a healing period of four weeks, bone blocks were embedded in LR White resin. Three consecutive sections per defect were analyzed as follows: two with light microscopy using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining and one with transmission electron microscopy. RESULTS Multinucleated giant cells appeared on both biomaterials. On BCP, MNGCs had a flat morphology and were not observed in resorption lacunae. On DBBM, the MNGCs appeared more round and were often found in shallow concavities. MNGCs on both biomaterials demonstrated a varying degree of TRAP staining, with a tendency toward higher staining intensity of MNGCs on BCP. At the ultrastructural level, signs of superficial dissolution of BCP together with phagocytosis of minor fragments were observed. MNGCs on the surface of DBBM demonstrated sealing zones and ruffled borders, both features of mature osteoclasts. CONCLUSION MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations.

Detection of dentinal cracks after root-end resection: an ex vivo study comparing microscopy and endoscopy with scanning electron microscopy

Dentinal cracks are occasionally observed at the cut root face after root-end resection in apical surgery. The objective of this ex vivo study was to evaluate and compare the efficiency of visual aids to identify root-end dentinal cracks.

Mechanism of action of tin-containing fluoride solutions as anti-erosive agents in dentine - an in vitro tin-uptake, tissue loss, and scanning electron microscopy study

Solutions containing tin and fluoride exhibit remarkable anti-erosive properties with tin ions as a major agent. To elucidate its mechanism of action in dentine, the tin uptake on and in the tissue was investigated and related to histological findings and substance loss. Samples were treated twice daily, each treatment lasting for 2 min, with fluoride solutions [pH 4.5; 1,500 parts per million (p.p.m.) F] containing 2,100, 1,400, or 400 p.p.m. Sn as SnCl(2). In experiments 1 and 2, samples were eroded with citric acid (pH 2.3) six times each day, each treatment lasting for 5 min; in experiment 2, the demineralized organic matrix was continuously digested by collagenase; in experiment 3, no erosive challenges were performed. Sample surfaces and cross-sections were investigated using energy dispersive X-ray spectroscopy, scanning electron microscopy, and profilometry. Surface retention of tin was found in almost all treatment groups and was highest in experiment 2. On cross-sections, tin was retained within the organic matrix; in mineralized areas, tin was found mainly within a depth of 10 mum. Test solutions inhibited substance loss significantly; in experiment 2, the effect was dose-dependent. Erosion inhibition seemed to depend mainly on the incorporation of tin in the mineralized dentine when the organic portion was preserved, but on surface precipitation when the organic portion was continuously digested.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Valve disease in chronic venous disorders: a quantitative ultrastructural analysis by transmission electron microscopy and stereology

INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 μm3/m2; in group B, 0.67 μm3/m2; in group C, 0.97 μm3/m2. The ratio was similar for collagen (A, 15.7 μm3/m2; B, 26.8 μm3/m2; C, 30.1 μm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Stereology meets electron tomography: towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung

BACKGROUND: Repeated bronchoalveolar lavage (BAL) has been used in animals to induce surfactant depletion and to study therapeutical interventions of subsequent respiratory insufficiency. Intratracheal administration of surface active agents such as perfluorocarbons (PFC) can prevent the alveolar collapse in surfactant depleted lungs. However, it is not known how BAL or subsequent PFC administration affect the intracellular and intraalveolar surfactant pool. METHODS: Male wistar rats were surfactant depleted by BAL and treated for 1 hour by conventional mechanical ventilation (Lavaged-Gas, n = 5) or partial liquid ventilation with PF 5080 (Lavaged-PF5080, n = 5). For control, 10 healthy animals with gas (Healthy-Gas, n = 5) or PF5080 filled lungs (Healthy-PF5080, n = 5) were studied. A design-based stereological approach was used for quantification of lung parenchyma and the intracellular and intraalveolar surfactant pool at the light and electron microscopic level. RESULTS: Compared to Healthy-lungs, Lavaged-animals had more type II cells with lamellar bodies in the process of secretion and freshly secreted lamellar body-like surfactant forms in the alveoli. The fraction of alveolar epithelial surface area covered with surfactant and total intraalveolar surfactant content were significantly smaller in Lavaged-animals. Compared with Gas-filled lungs, both PF5080-groups had a significantly higher total lung volume, but no other differences. CONCLUSION: After BAL-induced alveolar surfactant depletion the amount of intracellularly stored surfactant is about half as high as in healthy animals. In lavaged animals short time liquid ventilation with PF5080 did not alter intra- or extracellular surfactant content or subtype composition.

Reprint of "Stereology meets electron tomography: towards quantitative 3D electron microscopy" [J. Struct. Biol. 159 (2007) 443-450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections

We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.