Jean-Pierre Goudonnet

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.

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.

Analysis of image formation with a photon scanning tunneling microscope

The photon scanning tunneling microscope (PSTM ) is based on the frustration of a total internal reflected beam by the end of an optical fiber. Until now it has been used to obtain topographic information, generally for smooth samples. We report theoretical as well as experimental results on the observation of a step on a quartz substrate with the PSTM. These results demonstrate the effects on image formation of the distance between the fiber tip and the sample surface, the orientation of the incident beam with respect to the step, the polarization, and the coherence of the light. Good agreement exists between numerical simulations and experiments. We show that a perturbative approach, whose validity has been checked by comparison with a rigorous volume integral method, provides physical insight into the main features of the different images.

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.

Imaging of surface plasmon launch and propagation using a photon scanning tunneling microscope

Abstract The spectroscopic capability of the photon scanning tunneling microscope is exploited to study directly the launch and propagation of surface plasmons on thin silver films. Two input beams, of different wavelength, are incident through the prism in a prism-Ag film-air-fibre tip system. Both excite surface plasmons at the Ag-air interface and light of both wavelengths is coupled into the fibre probe via the respective surface plasmon evanescent fields. One laser beam is used for instrument control. The second, or probe beam is tightly focused on the sample, within the area of the unfocused or control beam, giving a well-defined and symmetrical, confined surface plasmon launch site. However, the image at the probe wavelength is highly asymmetrical in section with an exponential tail extending beyond one side of the launch site. This demonstrates in a very direct fashion the propagation of surface plasmons; a propagation length of ∼11.7 μm is measured at a probe wavelength of 543.5 nm. On rough Ag films the excitation of localised scattering centres is also observed in addition to the launch of delocalised surface plasmons.

Observation of optical waveguides by using a photon scanning tunneling microscope

The photon scanning tunneling microscope (PSTM) is based on the frustration of total internal reflection by the apex of an optical fiber placed near the surface of reflection. It has so far been used to obtain topographic information. This article shows that it also allows one to reach local variations of the refractive index of the sample. The PSTM, like many local probes, is operated on a constant intensity level. In that case a theoretical analysis based on a simple model shows that a change of the refractive index can actually be seen with the PSTM. The authors find that the corrugation height depends on the distance between the tip and the sample surface. The local refractive index of microguides diffused in glass has been studied experimentally with the PSTM. Relative variations in the order of 10-3 have been observed.

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.

Scanning tunnelling microscopy of 16S ribosomal RNA in water

The scanning tunnelling microscope has been used to image 16S ribosomal RNA molecules in water electrophoretically deposited on graphite surface. Two kinds of images have been obtained: images showing aggregates of 16S ribosomal RNA molecules similar to those obtained from DNA solutions and others showing individual 16S ribosomal RNA molecules. An interesting characteristic of these images, recorded in constant current mode, is that the 16S ribosomal RNA molecules appear to be located below the graphite surface. The morphology and several structural parameters of the molecules were consistent with the data obtained from electron microscopy.

Influence des contraintes sur les propriétés élastiques de surface du gypse sondées par microscopie à force atomique

Resume La modulation de force en microscopie a force atomique (AFM) permet de cartographier les inhomogeneites des proprietes elastiques de surface. Linfluence des contraintes laterales sur lelasticite de surface a pu etre mise en evidence par modulation de force. Letude a porte sur la face (010) du gypse. Au niveau atomique, les images topographiques different de celles sondant lelasticite de surface. Au niveau microscopique, les images delasticite dependent des contraintes exercees sur le cristal. Sans contrainte, la surface presente une elasticite uniforme. Cependant, les contraintes laterales revelent des phenomenes de glissement, de cisaillement et de nœuds delasticite.