Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles.

We use cryo-electron microscopy (cryo-EM) to study the 3D shapes of 94-bp-long DNA minicircles and address the question of whether cyclization of such short DNA molecules necessitates the formation of sharp, localized kinks in DNA or whether the necessary bending can be redistributed and accomplished within the limits of the elastic, standard model of DNA flexibility. By comparing the shapes of covalently closed, nicked and gapped DNA minicircles, we conclude that 94-bp-long covalently closed and nicked DNA minicircles do not show sharp kinks while gapped DNA molecules, containing very flexible single-stranded regions, do show sharp kinks. We corroborate the results of cryo-EM studies by using Bal31 nuclease to probe for the existence of kinks in 94-bp-long minicircles.

Atomic force microscopy of nucleoprotein complexes.

Recent data on the AFM studies of nucleoprotein complexes of different types are reviewed in this paper. The first section describes the progress in the sample preparation methods for AFM studies of nucleic acids and nucleoprotein complexes. The second part of this paper reviews AFM data on studies of complexes of DNA with regulatory proteins. These studies include two different types of DNA distortion induced by proteins binding: local bending of DNA at sites of protein binding and formation of large loops due to protein-protein interactions between molecules bound to distant sites along the DNA molecules (DNA looping). The prospects for use of AFM for physical mapping of genomes are discussed in this section as well. The third part of the paper reviews data on studies of complexes of DNA with non-sequence specific binding proteins. Special emphasis is given to studies of chromatin which have resulted in progress in the understanding of structure of native chromatin fiber. In this section, novel data on AFM studies of RecA-DNA filaments and complexes of dsRNA with the dsRNA-specific protein p25 are also presented. Discussion of the substrate preparation procedures in relation to the AFM studies of nucleoprotein complexes is given in the final section.

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.

Examination of Heterogeneous Crossing Sequences Between Toner and Rollerball Pen Strokes by Digital Microscopy and 3-D Laser Profilometry

The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner. With the mass marketing of laser printers and the popularity of rollerball pens, the determination of line crossing sequences between such instruments is encountered by forensic document examiners. This type of crossing presents difficulties with optical microscopic line crossing techniques involving ballpoint pens or gel pens and toner (1-4). Indeed, the rollerball's aqueous ink penetrates through the toner and is absorbed by the fibers of the paper, leaving the examiner with the impression that the toner is above the ink even when it is not (5). Novotny and Westwood (3) investigated the possibility of determining aqueous ink and toner crossing sequences by microscopic observation of the intersection before and after toner removal. A major disadvantage of their study resides in destruction of the sample by scraping off the toner line to see what was underneath. The aim of this research was to investigate the ways to overcome these difficulties through digital microscopy and three-dimensional (3-D) laser profilometry. The former was used as a technique for the determination of sequences between gel pen and toner printing strokes, but provided less conclusive results than that of an optical stereomicroscope (4). 3-D laser profilometry, which allows one to observe and measure the topography of a surface, has been the subject of a number of recent studies in this area. Berx and De Kinder (6) and Schirripa Spagnolo (7,8) have tested the application of laser profilometry to determine the sequence of intersections of several lines. The results obtained in these studies overcome disadvantages of other methods applied in this area, such as scanning electron microscope or the atomic force microscope. The main advantages of 3-D laser profilometry include the ease of implementation of the technique and its nondestructive nature, which does not require sample preparation (8-10). Moreover, the technique is reproducible and presents a high degree of freedom in the vertical axes (up to 1000 μm). However, when the paper surface presents a given roughness, if the pen impressions alter the paper with a depth similar to the roughness of medium, the results are not always conclusive (8). It becomes difficult in this case to distinguish which characteristics can be imputed to the pen impressions or the quality of the paper surface. This important limitation is assessed by testing different types of paper of variable quality (of different grammage and finishing) and the writing pressure. The authors will therefore assess the limits of 3-D laser profilometry technique and determine whether the method can overcome such constraints. Second, the authors will investigate the use of digital microscopy because it presents a number of advantages: it is efficient, user-friendly, and provides an objective evaluation and interpretation.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Microcontroller-driven fluid-injection system for atomic force microscopy.

We present a programmable microcontroller-driven injection system for the exchange of imaging medium during atomic force microscopy. Using this low-noise system, high-resolution imaging can be performed during this process of injection without disturbance. This latter circumstance was exemplified by the online imaging of conformational changes in DNA molecules during the injection of anticancer drug into the fluid chamber.

Label-free cytotoxicity screening assay by digital holographic microscopy.

Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca(2+)-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca(2+) from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.

Is it the grain size or the characteristic pore size that controls the induced polarization relaxation time of clean sands and sandstones?

There is a wide range of evidence to suggest that permeability can be constrained through of induced polarization measurements. For clean sands and sandstones, current mechanistic models of induced polarization predict a relationship between the low-frequency time constant inferred from induced polarization measurements and the grain diameter. A number of observations do, however, disagree with this and indicate that the observed relaxation behavior is rather governed by the so-called dynamic pore radius L. To test this hypothesis, we have developed a set of new scaling relationships, which allow the relaxation time to be computed from the pore size and the permeability to be computed from both the Cole-Cole time constant and the formation factor. Moreover, these new scaling relationships can be also used to predict the dependence of the Cole-Cole time constant as a function of the water saturation under unsaturated conditions. Comparative tests of the proposed new relationships with regard to various published experimental results for saturated clean sands and sandstones as well as for partially saturated clean sandstones, do indeed confirm that the dynamic pore radius L is a much more reliable indicator of the observed relaxation behavior than grain-size-based models.

Self construction, cognitive conflicts and polarization in bulimia nervosa

This study explores the cognitive structures, understood as construct systems, of patients suffering from bulimia nervosa (BN). Previous studies investigated the construct systems of disordered eaters suggesting that they had a higher distance between their construction of the self and the «ideal self», and also more rigidity. In addition to these aspects, this study explored the presence of implicative dilemmas (ID). Thirty two women who met criteria for BN and were treated in a specialized center were compared to a non clinical group composed by 32 women matched by age. All participants were assessed using Repertory Grid Technique (RGT). In BN patients it was more common (71.9%) to find IDs than in controls (18.8%). They also showed higher polarization and higher self-ideal discrepancies (even more for those with a long history of BN). The measures provided by the RGT can be useful for the assessment of self-construction and cognitive conflicts in BN patients and to appreciate their role in this disorder. In addition, this technique could be helpful for clinicians to explore the patient"s constructs system, and specially to identify IDs that could be maintaining the symptoms or hindering change in order to focus on them to facilitate improvement.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

3D solutions in transmission electron (Cryo) -microscopy in biology

One of the main problems in transmission electron microscopy in thebiological field is the tri-dimensionality. This article explains the technicalprocedures and requirements to prepare biological specimens preserving themclosest to their native state to perform 3D reconstruction of the macromolecularcomplexes and cellular structures in their natural environment.

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.

Atomic force microscopy: probing the nanoworld

Atomic Force Microscope and related techniques have played a key role in the development of the nanotechnology revolution that is taking place in science. This paper reviews the basic principles behind the technique and its different operation modes and applications, pointing out research worksperformed in the Nanometric Techniques Unit of the CCiTUB in order to exemplify the vast array of capabilities of these instruments.