Precession electron diffraction in the transmission electron Microscope: electron crystallography and orientational mapping

Precession electron diffraction (PED) is a hollow cone non-stationary illumination technique for electron diffraction pattern collection under quasikinematicalconditions (as in X-ray Diffraction), which enables “ab-initio” solving of crystalline structures of nanocrystals. The PED technique is recently used in TEMinstruments of voltages 100 to 300 kV to turn them into true electron iffractometers, thus enabling electron crystallography. The PED technique, when combined with fast electron diffraction acquisition and pattern matching software techniques, may also be used for the high magnification ultra-fast mapping of variable crystal orientations and phases, similarly to what is achieved with the Electron Backscatter Diffraction (EBSD) technique in Scanning ElectronMicroscopes (SEM) at lower magnifications and longer acquisition times.

Cryo-electron microscopy of vitreous sections of native biological cells and tissues : methodology and applications

Résumé L'eau est souvent considérée comme une substance ordinaire puisque elle est très commune dans la nature. En fait elle est la plus remarquable de toutes les substances. Sans l'eau la vie sur la terre n'existerait pas. L'eau représente le composant majeur de la cellule vivante, formant typiquement 70 à 95% de la masse cellulaire et elle fournit un environnement à d'innombrables organismes puisque elle couvre 75% de la surface de terre. L'eau est une molécule simple faite de deux atomes d'hydrogène et un atome d'oxygène. Sa petite taille semble en contradiction avec la subtilité de ses propriétés physiques et chimiques. Parmi celles-là, le fait que, au point triple, l'eau liquide est plus dense que la glace est particulièrement remarquable. Malgré son importance particulière dans les sciences de la vie, l'eau est systématiquement éliminée des spécimens biologiques examinés par la microscopie électronique. La raison en est que le haut vide du microscope électronique exige que le spécimen biologique soit solide. Pendant 50 ans la science de la microscopie électronique a adressé ce problème résultant en ce moment en des nombreuses techniques de préparation dont l'usage est courrant. Typiquement ces techniques consistent à fixer l'échantillon (chimiquement ou par congélation), remplacer son contenu d'eau par un plastique doux qui est transformé à un bloc rigide par polymérisation. Le bloc du spécimen est coupé en sections minces (d’environ 50 nm) avec un ultramicrotome à température ambiante. En général, ces techniques introduisent plusieurs artefacts, principalement dû à l'enlèvement d'eau. Afin d'éviter ces artefacts, le spécimen peut être congelé, coupé et observé à basse température. Cependant, l'eau liquide cristallise lors de la congélation, résultant en une importante détérioration. Idéalement, l'eau liquide est solidifiée dans un état vitreux. La vitrification consiste à refroidir l'eau si rapidement que les cristaux de glace n'ont pas de temps de se former. Une percée a eu lieu quand la vitrification d'eau pure a été découverte expérimentalement. Cette découverte a ouvert la voie à la cryo-microscopie des suspensions biologiques en film mince vitrifié. Nous avons travaillé pour étendre la technique aux spécimens épais. Pour ce faire les échantillons biologiques doivent être vitrifiés, cryo-coupées en sections vitreuse et observées dans une cryo-microscope électronique. Cette technique, appelée la cryo- microscopie électronique des sections vitrifiées (CEMOVIS), est maintenant considérée comme étant la meilleure façon de conserver l'ultrastructure de tissus et cellules biologiques dans un état très proche de l'état natif. Récemment, cette technique est devenue une méthode pratique fournissant des résultats excellents. Elle a cependant, des limitations importantes, la plus importante d'entre elles est certainement dû aux artefacts de la coupe. Ces artefacts sont la conséquence de la nature du matériel vitreux et le fait que les sections vitreuses ne peuvent pas flotter sur un liquide comme c'est le cas pour les sections en plastique coupées à température ambiante. Le but de ce travail a été d'améliorer notre compréhension du processus de la coupe et des artefacts de la coupe. Nous avons ainsi trouvé des conditions optimales pour minimiser ou empêcher ces artefacts. Un modèle amélioré du processus de coupe et une redéfinitions des artefacts de coupe sont proposés. Les résultats obtenus sous ces conditions sont présentés et comparés aux résultats obtenus avec les méthodes conventionnelles. Abstract Water is often considered to be an ordinary substance since it is transparent, odourless, tasteless and it is very common in nature. As a matter of fact it can be argued that it is the most remarkable of all substances. Without water life on Earth would not exist. Water is the major component of cells, typically forming 70 to 95% of cellular mass and it provides an environment for innumerable organisms to live in, since it covers 75% of Earth surface. Water is a simple molecule made of two hydrogen atoms and one oxygen atom, H2O. The small size of the molecule stands in contrast with its unique physical and chemical properties. Among those the fact that, at the triple point, liquid water is denser than ice is especially remarkable. Despite its special importance in life science, water is systematically removed from biological specimens investigated by electron microscopy. This is because the high vacuum of the electron microscope requires that the biological specimen is observed in dry conditions. For 50 years the science of electron microscopy has addressed this problem resulting in numerous preparation techniques, presently in routine use. Typically these techniques consist in fixing the sample (chemically or by freezing), replacing its water by plastic which is transformed into rigid block by polymerisation. The block is then cut into thin sections (c. 50 nm) with an ultra-microtome at room temperature. Usually, these techniques introduce several artefacts, most of them due to water removal. In order to avoid these artefacts, the specimen can be frozen, cut and observed at low temperature. However, liquid water crystallizes into ice upon freezing, thus causing severe damage. Ideally, liquid water is solidified into a vitreous state. Vitrification consists in solidifying water so rapidly that ice crystals have no time to form. A breakthrough took place when vitrification of pure water was discovered. Since this discovery, the thin film vitrification method is used with success for the observation of biological suspensions of. small particles. Our work was to extend the method to bulk biological samples that have to be vitrified, cryosectioned into vitreous sections and observed in cryo-electron microscope. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS). It is now believed to be the best way to preserve the ultrastructure of biological tissues and cells very close to the native state for electron microscopic observation. Since recently, CEMOVIS has become a practical method achieving excellent results. It has, however, some sever limitations, the most important of them certainly being due to cutting artefacts. They are the consequence of the nature of vitreous material and the fact that vitreous sections cannot be floated on a liquid as is the case for plastic sections cut at room temperature. The aim of the present work has been to improve our understanding of the cutting process and of cutting artefacts, thus finding optimal conditions to minimise or prevent these artefacts. An improved model of the cutting process and redefinitions of cutting artefacts are proposed. Results obtained with CEMOVIS under these conditions are presented and compared with results obtained with conventional methods.

Cryo-negative staining: advantages and applications for three-dimensional electron microscopy of biological macromolecules

Les échantillons biologiques ne s?arrangent pas toujours en objets ordonnés (cristaux 2D ou hélices) nécessaires pour la microscopie électronique ni en cristaux 3D parfaitement ordonnés pour la cristallographie rayons X alors que de nombreux spécimens sont tout simplement trop << gros D pour la spectroscopie NMR. C?est pour ces raisons que l?analyse de particules isolées par la cryo-microscopie électronique est devenue une technique de plus en plus importante pour déterminer la structure de macromolécules. Néanmoins, le faible rapport signal-sur-bruit ainsi que la forte sensibilité des échantillons biologiques natifs face au faisceau électronique restent deux parmi les facteurs limitant la résolution. La cryo-coloration négative est une technique récemment développée permettant l?observation des échantillons biologiques avec le microscope électronique. Ils sont observés à l?état vitrifié et à basse température, en présence d?un colorant (molybdate d?ammonium). Les avantages de la cryo-coloration négative sont étudiés dans ce travail. Les résultats obtenus révèlent que les problèmes majeurs peuvent êtres évités par l?utilisation de cette nouvelle technique. Les échantillons sont représentés fidèlement avec un SNR 10 fois plus important que dans le cas des échantillons dans l?eau. De plus, la comparaison de données obtenues après de multiples expositions montre que les dégâts liés au faisceau électronique sont réduits considérablement. D?autre part, les résultats exposés mettent en évidence que la technique est idéale pour l?analyse à haute résolution de macromolécules biologiques. La solution vitrifiée de molybdate d?ammonium entourant l?échantillon n?empêche pas l?accès à la structure interne de la protéine. Finalement, plusieurs exemples d?application démontrent les avantages de cette technique nouvellement développée.<br/><br/>Many biological specimens do not arrange themselves in ordered assemblies (tubular or flat 2D crystals) suitable for electron crystallography, nor in perfectly ordered 3D crystals for X-ray diffraction; many other are simply too large to be approached by NMR spectroscopy. Therefore, single-particles analysis has become a progressively more important technique for structural determination of large isolated macromolecules by cryo-electron microscopy. Nevertheless, the low signal-to-noise ratio and the high electron-beam sensitivity of biological samples remain two main resolution-limiting factors, when the specimens are observed in their native state. Cryo-negative staining is a recently developed technique that allows the study of biological samples with the electron microscope. The samples are observed at low temperature, in the vitrified state, but in presence of a stain (ammonium molybdate). In the present work, the advantages of this novel technique are investigated: it is shown that cryo-negative staining can generally overcome most of the problems encountered with cryo-electron microscopy of vitrified native suspension of biological particles. The specimens are faithfully represented with a 10-times higher SNR than in the case of unstained samples. Beam-damage is found to be considerably reduced by comparison of multiple-exposure series of both stained and unstained samples. The present report also demonstrates that cryo-negative staining is capable of high- resolution analysis of biological macromolecules. The vitrified stain solution surrounding the sample does not forbid the access to the interna1 features (ie. the secondary structure) of a protein. This finding is of direct interest for the structural biologist trying to combine electron microscopy and X-ray data. developed electron microscopy technique. Finally, several application examples demonstrate the advantages of this newly

Study of biomacromolecule conformational changes by Scanning Force Microscopy

L?objectif de ce travail de thèse est l?étude des changements conformationels des biomacromolecules à l?échelle d?une molécule unique. Pour cela on a utilisé la Microscopie à Force Atomique (AFM) appliqué à l?étude des protéines et des acides nucléiques déposés sur une surface. Dans ce type de microscopie, une pointe très fine attachée à l?extrémité d?un levier est balayée au dessus d?une surface. L?interaction de la pointe avec la surface de l?échantillon induit la déflection du levier et ce phénomène permet de reconstruire la topographie de l?échantillon. Très importante dans cette technique est la possibilité de travailler en liquide. Cela permet de étudier les biomolécules en conditions quasi-physiologiques sans qu?elles perdent leur activité. On a étudié GroEL, la chaperonin de E.coli, qui est un homo oligomère avec une structure à double anneau qui joue un rôle très important dans le repliement des protéines dénaturées et celles qui viennent d?être synthétisées. En particulier on a focalisé notre attention sur la stabilité mécanique et sur les changements conformationels qui ont lieu pendant l?activité de GroEL. Une analyse détaillée des changements dans la stabilité mécanique et des effets produits par la liaison et l?hydrolyse de l?ATP est présentée dans ce travail. On a montré que le point le plus faible dans la structure de GroEL est l?interface entre les deux anneaux et que l?étape critique dans l?affaiblissement de la structure est l?hydrolyse de l?ATP. En ce qui concerne le changement conformationel, le passage d?une surface hydrophobe à hydrophile, induit par l?hydrolyse de l?ATP, a été montré. Ensuite on a étudié le changement dans la conformation et dans la topologie de l?ADN résultant de l?interaction avec des molécules spécifiques et en réponse à l?exposition des cellules de E.coli à des conditions de stress. Le niveau de surenroulement est un paramètre très sensible, de façon variée, à tous ces facteurs. Les cellules qui ont crus à de températures plus élevées que leur température optimale ont la tendance à diminuer le nombre de surenroulements négatif pour augmenter la stabilité thermique de leur plasmides. L?interaction avec des agents intercalant induit une transition d?un surenroulement négatif à un surenroulement positif d?une façon dépendante de la température. Finalement, l?effet de l?interaction de l?ADN avec des surfaces différentes a été étudié et une application pratique sur les noeuds d?ADN est présentée.<br/><br/>The aim of the present thesis work is to study the conformational changes of biomacromolecules at the single molecule level. To that end, Atomic Force Microcopy (AFM) imaging was performed on proteins and nucleic acids adsorbed onto a surface. In this microcopy technique a very sharp tip attached at the end of a soft cantilever is scanned over a surface, the interaction of the tip with the sample?s surface will induce the deflection of the cantilever and thus it will make possible to reconstruct the topography. A very important feature of AFM is the possibility to operate in liquid, it means with the sample immersed in a buffer solution. This allows one to study biomolecules in quasi-physiological conditions without loosing their activity. We have studied GroEL, the chaperonin of E.coli, which is a double-ring homooligomer which pays a very important role in the refolding of unfolded and newly synthetized polypeptides. In particular we focus our attention on its mechanical stability and on the conformational change that it undergoes during its activity cycle. A detailed analysis of the change in mechanical stability and how it is affected by the binding and hydrolysis of nucleotides is presented. It has been shown that the weak point of the chaperonin complex is the interface between the two rings and that the critical step to weaken the structure is the hydrolysis of ATP. Concerning the conformational change we have directly measured, with a nanometer scale resolution, the switching from a hydrophobic surface to a hydrophilic one taking place inside its cavity induced by the ATP hydrolysis. We have further studied the change in the DNA conformation and topology as a consequence of the interaction with specific DNA-binding molecules and the exposition of the E.coli cells to stress conditions. The level of supercoiling has been shown to be a very sensitive parameter, even if at different extents, to all these factors. Cells grown at temperatures higher than their optimum one tend to decrease the number of the negative superhelical turns in their plasmids in order to increase their thermal stability. The interaction with intercalating molecules induced a transition from positive to negative supercoiling in a temperature dependent way. The effect of the interaction of the DNA with different surfaces has been investigated and a practical application to DNA complex knots is reported.<br/><br/>Observer les objets biologiques en le touchant Schématiquement le Microscope a Force Atomique (AFM) consiste en une pointe très fine fixée a l?extrémité d?un levier Lors de l?imagerie, la pointe de l?AFM gratte la surface de l?échantillon, la topographie de celui-ci induit des déflections du levier qui sont enregistrées au moyen d?un rayon laser réfléchi par le levier. Ces donnés sont ensuit utilisés par un ordinateur pour reconstituer en 3D la surface de l?échantillon. La résolution de l?instrument est fonction entre autre de la dureté, de la rugosité de l?échantillon et de la forme de la pointe. Selon l?échantillon et la pointe utilisée la résolution de l?AFM peut aller de 0.1 A (sur des cristaux) a quelque dizaine de nanomètres (sur des cellules). Cet instrument est particulierment intéressant en biologie en raison de sa capacité à imager des échantillons immergés dans un liquide, c?est à dire dans des conditions quasiphysiologiques. Dans le cadre de ce travail nous avons étudié les changements conformationels de molécules biologiques soumises à des stimulations externes. Nous avons essentielment concentré notre attention sur des complexes protéiques nommé Chaperons Moléculaires et sur des molécules d?ADN circulaire (plasmides). Les Chaperons sont impliqués entre autre dans la résistance des organismes vivants aux stress thermiques et osmotiques. Leur activité consiste essentielment à aider les autres protéines à être bien pliés dans leur conformation finale et, en conséquence, à eviter que ils soient dénaturées et que ils puissent s?agréger. L?ADN, quant à lui est la molécule qui conserve, dans sa séquence, l?information génétique de tous les organismes vivants. Ce travail a spécifiquement concerné l?étude des changements conformationels des chaperonins suit a leur activation par l?ATP. Ces travaux ont montrés a l?échelle de molécule unique la capacité de ces protéines de changer leur surface de hydrophobique a hydrophilique. Nous avons également utilisé l?AFM pour étudier le changement du nombre des surenroulements des molécules d?ADN circulaire lors d?une exposition à un changement de température et de force ionique. Ces travaux ont permis de montrer comment la cellule regle le nombre de surenroulements dans ces molécules pour répondre et contrôler l?expression génétique même dans de conditions extrêmes. Pour les deux molécules en général, c?était très important d?avoir la possibilité de observer leur transitions d?une conformation a l?autre directement a l?échelle d?une seul molécule et, surtout, avec une résolution largement au dessous des la longueur d?onde de la lumière visible que représente le limite pour l?imagerie optique.

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.

Characterisation of pigment particles by scanning electron microscope and image analysis programs

Coating and filler pigments have strong influence to the properties of the paper. Filler content can be even over 30 % and pigment content in coating is about 85-95 weight percent. The physical and chemical properties of the pigments are different and the knowledge of these properties is important for optimising of optical and printing properties of the paper. The size and shape of pigment particles can be measured by different analysers which can be based on sedimentation, laser diffraction, changes in electric field etc. In this master's thesis was researched particle properties especially by scanning electron microscope (SEM) and image analysis programs. Research included nine pigments with different particle size and shape. Pigments were analysed by two image analysis programs (INCA Feature and Poikki), Coulter LS230 (laser diffraction) and SediGraph 5100 (sedimentation). The results were compared to perceive the effect of particle shape to the performance of the analysers. Only image analysis programs gave parameters of the particle shape. One part of research was also the sample preparation for SEM. Individual particles should be separated and distinct in ideal sample. Analysing methods gave different results but results from image analysis programs corresponded even to sedimentation or to laser diffraction depending on the particle shape. Detailed analysis of the particle shape required high magnification in SEM, but measured parameters described very well the shape of the particles. Large particles (ecd~1 µm) could be used also in 3D-modelling which enabled the measurement of the thickness of the particles. Scanning electron microscope and image analysis programs were effective and multifunctional tools for particle analyses. Development and experience will devise the usability of analysing method in routine use.

Electric field calibration method for scanning electron microscope

This paper proposes a calibration method which can be utilized for the analysis of SEM images. The field of application of the developed method is a calculation of surface potential distribution of biased silicon edgeless detector. The suggested processing of the data collected by SEM consists of several stages and takes into account different aspects affecting the SEM image. The calibration method doesn’t pretend to be precise but at the same time it gives the basics of potential distribution when the different biasing voltages applied to the detector.

Electric field measurements using scanning electron microscope

The main idea of this diploma work is to study electric field distribution on the micro level. For this purpose a silicon edgeless detector was chosen as the object of investigation and scanning electron microscope as an investigation tool. Silicon edgeless detector is an important part of installation for studying proton-proton interactions in TOTEM experiment at Large Hadron Collider. For measurement of electric field distribution inside scanning electron microscope a voltage contrast method was applied.

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

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

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.

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.

Influence of the buffer environment on the Relative Biological Effectiveness of carbon ion radiation, investigated by Scanning Force Microscopy and gel electrophoresis

This work focuses on the analysis of the influence of environment on the relative biological effectiveness (RBE) of carbon ions on molecular level. Due to the high relevance of RBE for medical applications, such as tumor therapy, and radiation protection in space, DNA damages have been investigated in order to understand the biological efficiency of heavy ion radiation. The contribution of this study to the radiobiology research consists in the analysis of plasmid DNA damages induced by carbon ion radiation in biochemical buffer environments, as well as in the calculation of the RBE of carbon ions on DNA level by mean of scanning force microscopy (SFM). In order to study the DNA damages, besides the common electrophoresis method, a new approach has been developed by using SFM. The latter method allows direct visualisation and measurement of individual DNA fragments with an accuracy of several nanometres. In addition, comparison of the results obtained by SFM and agarose gel electrophoresis methods has been performed in the present study. Sparsely ionising radiation, such as X-rays, and densely ionising radiation, such as carbon ions, have been used to irradiate plasmid DNA in trishydroxymethylaminomethane (Tris buffer) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) environments. These buffer environments exhibit different scavenging capacities for hydroxyl radical (HO0), which is produced by ionisation of water and plays the major role in the indirect DNA damage processes. Fragment distributions have been measured by SFM over a large length range, and as expected, a significantly higher degree of DNA damages was observed for increasing dose. Also a higher amount of double-strand breaks (DSBs) was observed after irradiation with carbon ions compared to X-ray irradiation. The results obtained from SFM measurements show that both types of radiation induce multiple fragmentation of the plasmid DNA in the dose range from D = 250 Gy to D = 1500 Gy. Using Tris environments at two different concentrations, a decrease of the relative biological effectiveness with the rise of Tris concentration was observed. This demonstrates the radioprotective behavior of the Tris buffer solution. In contrast, a lower scavenging capacity for all other free radicals and ions, produced by the ionisation of water, was registered in the case of HEPES buffer compared to Tris solution. This is reflected in the higher RBE values deduced from SFM and gel electrophoresis measurements after irradiation of the plasmid DNA in 20 mM HEPES environment compared to 92 mM Tris solution. These results show that HEPES and Tris environments play a major role on preventing the indirect DNA damages induced by ionising radiation and on the relative biological effectiveness of heavy ion radiation. In general, the RBE calculated from the SFM measurements presents higher values compared to gel electrophoresis data, for plasmids irradiated in all environments. Using a large set of data, obtained from the SFM measurements, it was possible to calculate the survive rate over a larger range, from 88% to 98%, while for gel electrophoresis measurements the survive rates have been calculated only for values between 96% and 99%. While the gel electrophoresis measurements provide information only about the percentage of plasmids DNA that suffered a single DSB, SFM can count the small plasmid fragments produced by multiple DSBs induced in a single plasmid. Consequently, SFM generates more detailed information regarding the amount of the induced DSBs compared to gel electrophoresis, and therefore, RBE can be calculated with more accuracy. Thus, SFM has been proven to be a more precise method to characterize on molecular level the DNA damage induced by ionizing radiations.