Transmission electron microscopy in cell biology: sample preparation techniques and image information

Transmission electron microscopy is a proven technique in the field of cell biology and a very useful tool in biomedical research. Innovation and improvements in equipment together with the introduction of new technology have allowed us to improve our knowledge of biological tissues, to visualizestructures better and both to identify and to locate molecules. Of all the types ofmicroscopy exploited to date, electron microscopy is the one with the mostadvantageous resolution limit and therefore it is a very efficient technique fordeciphering the cell architecture and relating it to function. This chapter aims toprovide an overview of the most important techniques that we can apply to abiological sample, tissue or cells, to observe it with an electron microscope, fromthe most conventional to the latest generation. Processes and concepts aredefined, and the advantages and disadvantages of each technique are assessedalong with the image and information that we can obtain by using each one ofthem.

Advanced applications of scanning electron microscopy in geology

Nowadays Scanning Electron Microscopy (SEM) is a basic and fundamental tool in the study of geologic samples. The collision of a highlyaccelerated electron beam with the atoms of a solid sample results in theproduction of several radiation types than can be detected and analysed byspecific detectors, providing information of the chemistry and crystallography ofthe studied material. From this point of view, the chamber of a SEM can beconsidered as a laboratory where different experiments can be carried out. Theapplication of SEM to geology, especially in the fields of mineralogy andpetrology has been summarised by Reed (1996).The aim of this paper is to showsome recent applications in the characterization of geologic materials.

Biomedical and biological applications of scanning electron microscopy

This article summarizes the basic principles of scanning electron microscopy and the capabilities of the technique with different examples ofapplications in biomedical and biological research.

Scanning electron microscopy of the interaction between Cryptococcus magnus and Colletotrichum gloeosporioides on papaya fruit

The objective of this work was to investigate possible modes of action of the yeast Cryptococcus magnus in controlling anthracnose (Colletotrichum gloeosporioides) on post harvested papaya fruits. Scanning electron microscopy was used to analyze the effect of the yeast on inoculations done after harvest. Results showed that C. magnus is able to colonize wound surfaces much faster than the pathogen, outcompeting the later for space and probably for nutrients. In addition, C. magnus produces a flocculent matrix, which affects hyphae integrity. The competition for space and the production of substances that affect hyphae integrity are among the most important modes of action of this yeast.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼500nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: An electron microscopy and P-31 and C-13 nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using C-13- and P-31-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N-2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

New features of the layer-by-layer deposition of microcrystalline silicon films revealed by spectroscopic ellipsometry and high resolution transmission electron microscopy

Spectroscopic ellipsometry and high resolution transmission electron microscopy have been used to characterize microcrystalline silicon films. We obtain an excellent agreement between the multilayer model used in the analysis of the optical data and the microscopy measurements. Moreover, thanks to the high resolution achieved in the microscopy measurements and to the improved optical models, two new features of the layer-by-layer deposition of microcrystalline silicon have been detected: i) the microcrystalline films present large crystals extending from the a-Si:H substrate to the film surface, despite the sequential process in the layer-by-layer deposition; and ii) a porous layer exists between the amorphous silicon substrate and the microcrystalline silicon film.

Complementarity of radioautographic and immunohistochemical techniques for localizing neuroreceptors at the light and electron microscopy level

To assess relationships between neuropeptide-binding sites and receptor proteins in rat brain, the distribution of radioautographically labeled somatostatin and neurotensin-binding sites was compared to that of immunolabeled sst2A and NTRH receptor subtypes, respectively. By light microscopy, immunoreactive sst2A receptors were either confined to neuronal perikarya and dendrites or diffusely distributed in tissue. By electron microscopy, areas expressing somatodendritic sst2A receptors displayed only low proportions of membrane-associated, as compared to intracellular, receptors. Conversely, regions displaying diffuse sst2A labeling exhibited higher proportions of membrane-associated than intracellular receptors. Furthermore, the former showed only low levels of radioautographically labeled somatostatin-binding sites whereas the latter contained high densities of somatostatin-binding suggesting that membrane-associated receptors are preferentially recognized by the radioligand. In the case of NTRH receptors, there was a close correspondence between the light microscopic distribution of NTRH immunoreactivity and that of labeled neurotensin-binding sites. Within the substantia nigra, the bulk of immuno- and autoradiographically labeled receptors were associated with the cell bodies and dendrites of presumptive DA neurons. By electron microscopy, both markers were detected inside as well as on the surface of labeled neurons. At the level of the plasma membrane, their distribution was highly correlated and characterized by a lack of enrichment at the level of synaptic junctions and by a homogeneous distribution along the remaining neuronal surface, in conformity with the hypothesis of an extra-synaptic action of this neuropeptide. Inside labeled dendrites, there was a proportionally higher content of immunoreactive than radiolabeled receptors. Some of the immunolabeled receptors not recognized by the radioligand were found in endosome-like organelles suggesting that, as in the case of sst2A receptors, they may have undergone endocytosis subsequent to binding to the endogenous peptide

Electron microscopy of glial cells of the central nervous system in the crab Ucides cordatus

Invertebrate glial cells show a variety of morphologies depending on species and location. They have been classified according to relatively general morphological or functional criteria and also to their location. The present study was carried out to characterize the organization of glial cells and their processes in the zona fasciculata and in the protocerebral tract of the crab Ucides cordatus. We performed routine and cytochemical procedures for electron microscopy analysis. Semithin sections were observed at the light microscope. The Thiéry procedure indicated the presence of carbohydrates, particularly glycogen, in tissue and in cells. To better visualize the axonal ensheathment at the ultrastructural level, we employed a method to enhance the unsaturated fatty acids present in membranes. Our results showed that there are at least two types of glial cells in these nervous structures, a light one and a dark one. Most of the dark cell processes have been mentioned in the literature as extracellular matrix, but since they presented an enveloping membrane, glycogen and mitochondria - intact and with different degrees of disruption - they were considered to be glial cells in the present study. We assume that they correspond to the perineurial cells on the basis of their location. The light cells must correspond to the periaxonal cells. Some characteristics of the axons such as their organization, ensheathment and subcellular structures are also described.

Studies of the small bowel surface by scanning electron microscopy in infants with persistent diarrhea

We describe the ultrastructural abnormalities of the small bowel surface in 16 infants with persistent diarrhea. The age range of the patients was 2 to 10 months, mean 4.8 months. All patients had diarrhea lasting 14 or more days. Bacterial overgrowth of the colonic microflora in the jejunal secretion, at concentrations above 10(4) colonies/ml, was present in 11 (68.7%) patients. The stool culture was positive for an enteropathogenic agent in 8 (50.0%) patients: for EPEC O111 in 2, EPEC O119 in 1, EAEC in 1, and Shigella flexneri in 1; mixed infections due to EPEC O111 and EAEC in 1 patient, EPEC O119 and EAEC in 1 and EPEC O55, EPEC O111, EAEC and Shigella sonnei in 1. Morphological abnormalities in the small bowel mucosa were observed in all 16 patients, varying in intensity from moderate 9 (56.3%) to severe 7 (43.7%). The scanning electron microscopic study of small bowel biopsies from these subjects showed several surface abnormalities. At low magnification (100X) most of the villi showed mild to moderate stunting, but on several occasions there was subtotal villus atrophy. At higher magnification (7,500X) photomicrographs showed derangement of the enterocytes; on several occasions the cell borders were not clearly defined and very often microvilli were decreased in number and height; in some areas there was a total disappearance of the microvilli. In half of the patients a mucus-fibrinoid pseudomembrane was seen partially coating the enterocytes, a finding that provides additional information on the pathophysiology of persistent diarrhea.

Ultrastructural changes of Fusobacterium nucleatum as a defense mechanism against human neutrophil peptide-1 - A Transmission Electron Microscopy (TEM) study

Aims: The aim of this work was to assess the ultrastructural changes, cellular proliferation, and the biofilm formation ability of F. nucleatum as defense mechanisms against the effect of HNP-1. Materials and methods: The type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were cultured and incubated with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml) and one control group (0 µg/ml). Bacterial pellets from each concentration were processed for TEM imaging. Planktonic growth was assessed and colony forming units (CFU) were measured to determine the cellular proliferation. Scrambled HNP-1 was used for confirmation. Results: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. For ATCC 25586, the treated bacterial cells contained higher numbers of intracellular granules with increasing the peptide concentration. Planktonic growth of the two clinical strains were significantly enhanced (P<0.001) with gradually increased concentrations of HNP-1. None of the planktonic growth results of the 3 strains incubated with the scrambled HNP-1 was statistically significant. HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 and 9508, significantly (P<0.01 and P<0.001; respectively). Conclusions: The present in vitro study demonstrates that F. nucleatum has the ability to withstand the lethal effects of HNP-1 even at concentrations simulating the diseased periodontium in vivo. The increase in planktonic growth could act as defense mechanisms of F. nucleatum against HNP-1.

Étude structurale conformationnelle des toxines de l’anthrax par cryo-microscopie et dynamique moléculaire

Les toxines de l’anthrax font partie de la famille des toxines A-B dans laquelle la moitié B se fixe à la membrane de la cellule permettant par la suite la translocation de la moitié A. Dans le cas de l’anthrax, la moitié B est représentée par le Protective Antigen (PA) et la moitié A par les deux protéines Edema Factor (EF) et Lethal Factor (LF). Après le recrutement par les récepteurs cellulaires (CMG2 et TEM8), PA s’organise en heptamère. Il peut fixer jusqu'à 3 ligands (EF et LF) avant d'être endocyté. Les modèles actuels de PA suggèrent que la baisse de pH à l’intérieur des endosomes permet un changement de conformation de la forme pré-pore vers la forme pore et que les ligands EF et LF passeraient au travers le pore pour entrer dans le cytoplasme. Cependant, le diamètre du pore est environ dix fois inférieur à celui des ligands (10 Å contre 100 Å). Un processus de folding/unfolding a été proposé mais demeure controversé. Afin d'identifier le processus de passage des facteurs EF et LF dans le cytoplasme, nous avons déterminé par cryo-microscopie électronique combinée avec l’analyse d’image les structures tridimensionnelles des complexes formés par PA et LF aux étapes prépore et pore. Par la suite, une étude complémentaire par dynamique moléculaire nous a permis de modéliser à haute résolution les différentes interactions qui ont lieu au sein du complexe. La structure 3D du complexe prépore combiné à 3 LF a été déterminée à une résolution de 14 Å. Nous avons aussi calculé une structure préliminaire du complexe pore également combiné à 3 LF Celles-ci n’ont jamais été résolues auparavant et leur connaissance permet d’envisager l’étude en profondeur du mécanisme infectieux de l’Anthrax in vivo.

Applying scanning electron microscopy for the ultrastructural and clinical analysis of periprosthetic capsules in implant-based breast reconstruction

La reconstruction en deux étapes par expanseur et implant est la technique la plus répandue pour la reconstruction mammmaire post mastectomie. La formation d’une capsule périprothétique est une réponse physiologique universelle à tout corps étranger présent dans le corps humain; par contre, la formation d’une capsule pathologique mène souvent à des complications et par conséquent à des résultats esthétiques sous-optimaux. Le microscope électronique à balayage (MEB) est un outil puissant qui permet d’effectuer une évaluation sans pareille de la topographie ultrastructurelle de spécimens. Le premier objectif de cette thèse est de comparer le MEB conventionnel (Hi-Vac) à une technologie plus récente, soit le MEB environnemental (ESEM), afin de déterminer si cette dernière mène à une évaluation supérieure des tissus capsulaires du sein. Le deuxième objectif est d‘appliquer la modalité de MEB supérieure et d’étudier les modifications ultrastructurelles des capsules périprothétiques chez les femmes subissant différents protocoles d’expansion de tissus dans le contexte de reconstruction mammaire prothétique. Deux études prospectives ont été réalisées afin de répondre à nos objectifs de recherche. Dix patientes ont été incluses dans la première, et 48 dans la seconde. La modalité Hi-Vac s’est avérée supérieure pour l’analyse compréhensive de tissus capsulaires mammaires. En employant le mode Hi-Vac dans notre protocole de recherche établi, un relief 3-D plus prononcé à été observé autour des expanseurs BIOCELL® dans le groupe d’approche d’intervention retardée (6 semaines). Des changements significatifs n’ont pas été observés au niveau des capsules SILTEX® dans les groupes d’approche d’intervention précoce (2 semaines) ni retardée.