[CHARACTERIZATION OF THE DIFFERENCE IN THE MORPHOLOGY OF DETERGENT RESISTANT MEMBRANES DOMAINS ISOLATED FROM DIFFERENT CELL TYPES BY ATOMIC FORCE MICROSCOPY].

Planar raft and caveolae are specific membrane clusters with high concentration of cholesterol and lipids with saturated fatty acid. These clasters are resistant to detergents and are denoted as detergent resistant membranes domains (DRMs). Their morphology and size have been studied by atomic force microscopy. The size of planar rafts isolated by Librol from monocytes of healthy volunteers was 150.6 ± 68.6 nm--diameters and 5.7 ± 2.9 nm--height, the size of caveolae was 87.3 ± 46.1 nm--diameters and 9.4 ± 5.4 nm--height. Significant difference have been found morphology and size of DRMs isolated from monocytes of healthy volunteers and patients suffering from myocardial infarction as well as between DRMs isolated from endothelial cells. The study of time-dependent changes in the morphology of isolated planar rafts and caveolae has shown that they quickly aggregate during keeping. Therefore, to asses the actual size and morphology of the DRMS, they should be investigated immediately after isolation.

Mode-synthesizing atomic force microscopy for volume characterization of mixed metal nanoparticles.

Atomic force microscopy (AFM) and other techniques derived from AFM have revolutionized the understanding of materials and biology at the nanoscale, but mostly provide surface properties. The observation of subsurface nanoscale features and properties remains a great challenge in nanometrology. The operating principle of the mode-synthesizing AFM (MSAFM) is based on the interaction of two ultrasonic waves, one launched by the AFM probe fp , a second launched by the sample fs , and their resulting nonlinear frequency mixing. Recent developments highlighted the need for quantitative correlation between the role of the frequency actuation of the probe fp and the sample fs . Here we present the great potential of MSAFM for advanced volume characterization of metallic nanoparticles presenting a multilayered structure composed of a nickel core surrounded by a gold envelope.

Homogeneous large-scale crystalline nanoparticle-covered substrate with high SERS performance.

This article details the surface-enhanced Raman scattering (SERS) performance of plasmonic substrates fabricated by a physical metal evaporation technique that uses no precursor or intermediate coating. We outline a cost-effective nanofabrication protocol that uses common laboratory equipment to produce homogeneously covered crystalline nanoparticle substrates. Our fabrication yields a homogeneous SERS response over the whole surface. The platform is tested with methylene blue diluted at various concentrations to estimate the sensitivity, homogeneity, and reproducibility of the process. The capacity of the substrates is also confirmed with spectroscopic investigations of human microsomal cytochrome b5.

Impact of optical and structural aging in As2S3 microstructured optical fibers on mid-infrared supercontinuum generation.

We analyze optical and structural aging in As2S3 microstructured optical fibers (MOFs) that may have an impact on mid-infrared supercontinuum generation. A strong alteration of optical transparency at the fundamental OH absorption peak is measured for high-purity As2S3 MOF stored in atmospheric conditions. The surface evolution and inherent deviation of corresponding chemical composition confirm that the optical and chemical properties of MOFs degrade upon exposure to ambient conditions because of counteractive surface process. This phenomenon substantially reduces the optical quality of the MOFs and therefore restrains the spectral expansion of generated supercontinuum. This aging process is well confirmed by the good matching between previous experimental results and the reported numerical simulations based on the generalized nonlinear Schrödinger equation.

From surface to intracellular non-invasive nanoscale study of living cells impairments.

Among the enduring challenges in nanoscience, subsurface characterization of living cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale. However, measurements in liquid environments remain complex, in particular in the subsurface domain. Here we introduce liquid-mode synthesizing atomic force microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach, l-MSAFM, for nanoscale imaging of living cell in their physiological environment or in presence of a chemical stress agent could resolve the loss of inner structures induced by glyphosate, the main component of a well-known pesticide (RoundUp™). This firsthand ability to monitor the cell's inner response to external stimuli non-destructively and in liquid, has the potential to unveil critical nanoscale mechanisms of life science.

Label-free sensing and atomic force spectroscopy for the characterization of protein-DNA and protein-protein interactions: application to estrogen receptors.

In this paper we describe a new surface plasmon resonance (SPR) biosensor dedicated to potential estrogenic compounds prescreening, by developing an estrogen receptor (ER) specific DNA chip. Through the covalent binding of a DNA strain wearing the estrogen response element (ERE) to an activated 6-mercapto-1-hexadecanoic acid and 11-mercapto-1-undecanol self-assembled monolayer on gold surface, the SPR biosensor allows to detect specifically, quickly, and without any labeling the binding of ER in the presence of estrogen. In parallel, we investigated the ER interaction with itself, in order to study the formation of ER dimer apparently needed to activate the gene expression through ERE interaction. For that, we engaged force spectroscopy experiments that allowed us to prove that ER needs estrogen for its dimerization. Moreover, these ER/ER intermolecular measurements enabled to propose an innovative screening tool for anti-estrogenic compounds, molecules of interest for hormono-dependent cancer therapy.

Shear force microscopy with a nanoscale resolution.

This paper presents a shear force microscope having a nanometric resolution at high scan rates. Current techniques were reviewed and tested, and a design based on the use of a tuning fork is described. The use of a low quality factor enabled us to decrease the response time and increase the stability of the tracking. The microscope was coupled with a tunneling current detection, in order to study the interactions between the sample and the probe during scanning. As an example, a sharp nickel nanotip was used to image a gold surface, showing details down to a few nanometers, even at scanning rates of 4Hz.

Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy.

Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view. The objective of this work was to further describe this mechanism. Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions. The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24h after elicitation. In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells. After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains. These expansions were not observed on untreated grapevine cells. The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties. The elasticity is diminished in UV-treated cells. In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response. These results demonstrate cell wall strengthening by UV stress.

Observation of the posterior endothelial surface of the rabbit cornea using atomic force microscopy.

PURPOSE: To study the surface of normal corneal endothelium by means of atomic force microscopy (AFM). METHODS: The central corneal endothelial posterior surface of New Zealand white rabbits was examined. Specimens were observed in Balanced Salt Solution using the contact mode of the AFM either fresh or after fixation in cacodylate-buffered glutaraldehyde solution. Removal of sialic acid residues and hyaluronic acid was achieved by means of enzymatic treatment with neuraminidase and hyaluronidase. RESULTS: Observation of the fresh specimens revealed the presence of an apical endothelial surface coating material (glycocalyx). Removal of sialic acid residues and hyaluronic acid after enzymatic treatment using neuraminidase and hyaluronidase, respectively, permitted the elucidation of the structure of the nondigested coating material. Fixation of the samples resulted in removal of the surface coating material. The imaging of the fixed endothelium surface revealed the mosaic of polygonal cells with the apical flaps of cell junctions emerging over the cell surface. The cell shape and the other characteristics of the posterior surface fixed endothelium were comparable to those described in the literature using scanning electron microscopy. The scanning of very small ranges has provided high-resolution images at the nanometer level in fixed and fresh corneal endothelial surfaces. CONCLUSION: The atomic force microscope represents a new powerful imaging tool permitting high-resolution observation of corneal endothelium surface in fresh and minimally prepared fixed specimens.

Affinity scale between a carrier and a drug in DPI studied by atomic force microscopy.

The dry powder inhalers (DPIs) consist, in the most cases, of ordered mixture where the particles adhesion results of interactions between the drug and the carrier. Generally, one step of production process is the micronization of the drug particles in order to reduce the size for ordered mixing optimization. But this operation is known to partially create an amorphous surface. In this case, surrounding storage conditions, like relative humidity (RH), are able to modify the percentage of amorphous drug surface. The aim of this study was to investigate surface reactivity, surface energy and direct force measurements by atomic force microscopy (AFM) between lactose (carrier) and zanamivir (drug) crystals references in various conditions of RH. Secondly, an amorphization of the drug surface was induced by humidity relative treatment in order to evaluate the consequences of the transition from crystal to amorphous phase. The study demonstrated that the amorphization of drug surface induces an increase of drug affinity with the carrier surface. Ex situ and in situ amorphization of zanamivir tend to reach the affinity measured between raw materials: carrier and micronized drug particles. AFM allowed adhesion force discrimination between the different forms of the drug particles and demonstrated the potential for investigating adhesion properties in DPI formulation.

Dry powder inhaler: influence of humidity on topology and adhesion studied by AFM.

In the dry powder inhalers (DPIs), the adhesion results of the interactions between the active substance and the excipient. The carrier and the micronized drug particle morphologies are believed to affect the delivery of the drug. In this work, the couple studied was the lactose monohydrate and micronized zanamivir, used for the treatment of influenza. In a first approach, observations by scanning electron microscopy (SEM) have shown that the relative humidity (RH) greatly influenced the zanamivir amount fixed on the lactose monohydrate surface. This paper deals with the direct measurement in controlled atmosphere by atomic force microscopy (AFM) of the forces and the interaction ranges between a zanamivir probe and a lactose substrate. Selected zanamivir crystals were attached to the standard AFM probe. Different RH have been used in order to determine influent parameters permitting to identify the nature of adhesion forces between them. This study demonstrated that the increase of RH modified progressively the surface topology of the two components and increased the adhesion force.

Investigation by atomic force microscopy of forces at the origin of cement cohesion.

In cement paste, the cohesion results of the interactions between calcium silicate hydrate (CSH) surfaces in an interstitial ionic solution. (N, V, T) Monte Carlo simulations show that the interactions are due to the ion correlation forces influenced by the surface charge density, the ionic concentration and the ion valence. This paper deals with the direct measurement in solutions by atomic force microscopy (AFM) of the forces and the interaction ranges between a probe and an atomically smooth substrate covered by CSH nanoparticles. Different electrolytic solutions (Ca(OH)2, CaCl2, NaCl, NaOH) have been used in order to determine influent parameters permitting to identify the nature of acting forces. Investigations have been rendered possible by selecting appropriate experimental setup and solutions. The selected probe and substrate on which CSH nanoparticles have previously grown are neutral regarding the reactivity during experiments permitting the exchange of solutions. Results show that a force originates from electrostatic nature and differs from Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Agreement is found between experiments and (N,V,T) Monte Carlo simulations of ionic correlation forces. These forces are at the origin of the cohesion of cement paste.

Measuring magnetic susceptibilities of nanogram quantities of materials using microcantilevers.

We describe a novel technique for measuring magnetic susceptibilities of nanogram quantities of magnetic materials that utilizes the extreme force sensitivity of microcantilevers. The magnetic force acting on samples attached to the free end of a cantilever can be measured as changes in the resonance response of the cantilever. The shift in resonance frequency of the cantilever is proportional to the field gradient, whereas the deflection of a cantilever is proportional to the magnetic force. The magnetic susceptibility measurement is based on comparison of the forces acting on the sample and a reference material in the same magnetic field and field gradient. We have determined the magnetic susceptibilities of nanogram quantities of many paramagnetic materials. The measured magnetic susceptibilities show excellent agreement with values found in the literature.

In situ imaging of detergent-resistant membranes by atomic force microscopy.

Purified detergent-resistant membranes (DRMs) are powerful tools for the biochemical study of plasma membrane domains. To what extent these isolated DRMs correspond to native membrane domains remains, however, a matter of debate. The most immediate question to be answered concerns the in situ size range of DRMs, a determination that escapes classical microscopy techniques. In this study we show that in situ three-dimensional images of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tapping mode atomic force microscopy. These images establish that, prior to the isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15-20 microm(2). This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomains of living cells, which strongly suggests that membrane microdomains rearrange into larger DRMs during Triton X-100 treatment. Concomitantly, the images also reveal the presence of the cytoskeleton, which is resistant to detergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton.

The use of atomic force microscopy for the observation of corneal epithelium surface.

PURPOSE: To evaluate the feasibility of imaging normal corneal epithelium by means of atomic force microscopy (AFM). METHODS: Twelve normal corneas from six albino rabbits were examined using a commercial atomic force microscope. Six corneas were examined in balanced salt solution after fixation in glutaraldehyde 2.5% and six without any fixation. Rectangular silicon nitride cantilevers with a spring constant of 10 to 20 mN/m were used. The measured forces after imaging were less than 100 pN. All reported images were made with 512x512-pixel definition with typical scan rates ranging from 1 to 5 Hz. RESULTS: High-quality images of corneal epithelium surface were obtained from fixed and unfixed specimens in magnifications ranging from x2000 to x2,000,000. Imaging of fixed specimens was always easier. In unfixed specimens fuzzy images were very common, probably because of the presence of the cell glycocalyx. AFM revealed the typical polygonal corneal epithelial cells. The cell surface was covered by microprojections; at cell borders the microprojections were arranged in two characteristic parallel rows. Craterlike formations were revealed in several specimens. The microprojections' morphology and their surface details were revealed using magnifications up to x2,000,000. Three-dimensional representation of the images facilitated better understanding of the surface topography. Measurements in horizontal and vertical plane were made using the section analysis tool. CONCLUSIONS: In this work the AFM parameters appropriate for corneal epithelium imaging in physiological medium were defined. AFM represents a new powerful tool for corneal epithelium imaging, and its application in this field warrants further investigation.

Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal.

Difficulties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous buffer. A simple procedure that drastically enhances the successful imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a deflection signal, concomitant with the damping of the amplitude that can be followed by amplitude-distance curves. For intact cells, the evolution of the deflection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in finding the appropriate tapping conditions, the deflection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new field of applications for the atomic force microscope.

Detection of peptide-lipid interactions in mixed monolayers, using isotherms, atomic force microscopy, and fourier transform infrared analyses.

To improve the understanding of the membrane uptake of an amphipathic and positively charged vector peptide, we studied the interactions of this peptide with different phospholipids, the nature of whose polar headgroups and physical states were varied. Three lipids were considered: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG). The approach was carried out by three complementary methods: compression isotherms of monolayers and atomic force microscopy observations associated with Fourier transform infrared investigations. From analysis of the compression isotherms, it was concluded that the peptide interacts with all lipids and with an expansion of the mean molecular area, implying that both components form nonideal mixtures. The expansion was larger in the case of DOPG than for DPPC and DPPG because of an alpha to beta conformational transition with an increase in the peptide molar fraction. Atomic force microscopy observations showed that the presence of small amounts of peptide led to the appearance of bowl-like particles and that an increase in the peptide amounts generated the formation of filaments. In the case of DOPG, filaments were found at higher peptide molar fractions than already observed for DOPC because of the presence of negatively charged lipid headgroups.

Lipid-induced organization of a primary amphipathic peptide: a coupled AFM-monolayer study.

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a beta-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate.

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.

Atomic force microscopy of renal cells: limits and prospects.

In this brief review, we present three-dimensional images of living Madin-Darby canine kidney (MDCK) cells and CV-1 cells that illustrate the possibilities and limits in the use of atomic force microscopy (AFM) for studying the topography of the cell surfaces and of isolated biological membranes. We show that microvilli can be imaged at the surface of living epithelial cells. However, when these microvilli are abundant and close to each other, the geometry of the AFM tip only allows an access to the upper part of the structures and precludes nanometer range imaging of the cell surface. Such a nanometer range imaging was obtained with other cell types like CV-1 cells and with isolated biological membranes. It reveals that protruding particles 5 to 60 nm xy size, likely corresponding to membranes proteins, occupy most of the membrane surface. These images indicate that the AFM already gives an access to the cell surface structure at the mesoscopic scale, which constitutes a major step for the understanding of the structure-function relationships in membranes. Perspectives for a further step, the imaging at molecular resolution of membranes, are discussed.