Eric Lesniewska

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.

Study of C-S-H growth on C3S surface during its early hydration

A study to understand and quantify the growth mode of C-S-H during the hydration of tricalcium silicate grains is presented here based upon number of complementary approaches: study of C-S-H nucleation during the hydration of tricalcium silicate compared with experimental studies of homogeneous nucleation; experimental kinetics studies of C-S-H formation during tricalcium silicate hydration in dilute suspensions; direct observations of C-S-H growth by Atomic Force Microscopy; Simulation of curves obtained from experiments in dilute suspensions from a model based on AFM observations. With AFM the formation of CSH is observed by the agglomeration of identical elements 60×30×5 nm3 in size on alite surface. This agglomeration takes place perpendicularly and parallel to the surface. Numerical simulation of the experimental curves of the degree of hydration versus time enabled us to quantify the variation of the growth rates parallel and perpendicular to the surface with lime concentration. It is shown that the growth rate of C-S-H only depends on the lime concentration in solution.RésuméUne étude pour comprendre et mesurer le mode de croissance de C-S-H pendant lhydration des grains de silicate tricalcique est présentée ici, basée sur un certain nombre dapproches complémentaires: létude de la nucléation de C-S-H pendant lhydration du silicate tricalcique comparée aux études expérimentales de nucléation homogène; études expérimentales de cinétique de formation de C-S-H pendant lhydration du silicate tricalcique dans les suspensions diluées; observations directes de croissance de C-S-H par microscopie de Force Atomique; simulation des courbes obtenues à partir des expériences dans les suspensions diluées à partir dun modèle basé sur des observations AFM. LAFM permet dobserver la formation de CSH par lagglomération déléments de tailles identiques de 60×30×5 nm3 sur la surface dalite. Cette agglomération a lieu perpendiculairement et parallèlement à la surface. La simulation numérique des courbes expérimentales du degré dhydration en fonction du temps nous a permis de mesurer la variation des taux de croissance parallèle et perpendiculaire à la surface avec la concentration en chaux. On montre que le taux de croissance le taux de C-S-H dépend seulement de la concentration en chaux en solution.

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.

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.

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

Abstract. 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 β-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.

Investigations of surface forces between gypsum crystals in electrolytic solutions using microcantilevers

This paper introduces a new approach to the study of the interactions between gypsum faces in electrolytic solutions of calcium sulfate. A systematic study with respect to the orientation of crystals, the concentration of the solution, the type of electrolyte, and the duration time of the contact leads to an improved understanding of both the DLVO theory and the mechanism involved in the development of the ionic correlation force. Using an atomic force microscope, direct local measurement of forces between crystals defines the ideal conditions favorable to the adhesion. The most important factor in crystal coagulation was found to be the effective surface charge of each face.

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.

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.

Tapping Mode Atomic Force Microscopy allows the in situ Imaging of Fragile Membrane Structures and of Intact Cells Surface at High Resolution

Because of its potential applications in the understanding of membrane function, high resolution in situ imaging by AFM of the surface of cells in their aqueous medium remains an important goal. Based on two examples, the in situ imaging of detergent-resistant plasma membrane domains and of the lateral membrane of outer hair cells from the inner ear, we show that low-force tapping mode AFM allows the access to the topography of very fragile membrane structures and that details of a cell can be acquired with a lateral resolution of about 5 nm.