Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy.

Red blood cell (RBC) membrane fluctuations provide important insights into cell states. We present a spatial analysis of red blood cell membrane fluctuations by using digital holographic microscopy (DHM). This interferometric and dye-free technique, possessing nanometric axial and microsecond temporal sensitivities enables to measure cell membrane fluctuations (CMF) on the whole cell surface. DHM acquisition is combined with a model which allows extracting the membrane fluctuation amplitude, while taking into account cell membrane topology. Uneven distribution of CMF amplitudes over the RBC surface is observed, showing maximal values in a ring corresponding to the highest points on the RBC torus as well as in some scattered areas in the inner region of the RBC. CMF amplitudes of 35.9+/-8.9 nm and 4.7+/-0.5 nm (averaged over the cell surface) were determined for normal and ethanol-fixed RBCs, respectively.

Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy.

We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy.

The authors have developed a live-cell multimodality microscope combining epifluorescence with digital holographic microscopy; it has been implemented with a decoupling procedure allowing to separately measure from the quantitative phase important cell parameters including absolute volume, shape and integral intracellular refractive index. In combination with the numerous different specific fluorescent cellular probes, this multimodality microscopy can address important issues in cell biology. This is demonstrated by the study of intracellular calcium homeostasis associated with the change in cell volume, which play a critical role in the excitotoxicity-induced neuronal death.

Axial sub-nanometer accuracy in digital holographic microscopy

We present state-of-the-art dual-wavelength digital holographic microscopy (DHM) measurement on a calibrated 8.9 nm high chromium thin step sample and demonstrate sub-nanometer axial accuracy. By using a modified DHM reference calibrated hologram (RCH) reconstruction method, a temporal averaging procedure and a specific dual-wavelength DHM arrangement, it is shown that specimen topography can be measured with an accuracy, defined as the axial standard deviation, reduced to at least 0.9 nm. Indeed for the first time to the best of our knowledge, it is reported that averaging each of the two wavefronts recorded with real-time dual-wavelength DHM can provide up to 30% spatial noise reduction for the given configuration. Moreover, the presented experimental configuration achieves a temporal stability below 0.8 nm, thus paving the way to Angström range for dual-wavelength DHM.

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.