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.

Light field propagation by metal micro- and nanostructures.

The ability to sustain plasmon oscillations gives rise to unique properties of metal nanostructures, which can be exploited for the controlled manipulation of light fields on the nanoscale. In this context we investigate electromagnetic coupling effects within lithographically produced ensembles of gold nanoparticles with a photon scanning tunnelling microscope. To provide an interface between these nano-optical devices and classical far-field optics, we investigate surface plasmon propagation on microstructured metal thin films.

Direct interpretation of near-field optical images.

The interpretation of the detection process in near-field optical microscopy is reviewed on the basis of a discussion about the possibility of establishing direct comparisons between experimental images and the solutions of Maxwell equations or the electromagnetic local density of states. On the basis of simple physical arguments, it is expected that the solutions of Maxwell equations should agree with images obtained by collecting mode near-field microscopes, while the electromagnetic local density of states should be considered to provide a practical interpretation of illumination mode near-field microscopes. We review collecting mode near-field microscope images where the conditions to obtain good agreement with the solutions of Maxwell equations have indeed been identified. In this context of collecting mode near-field microscopes, a fundamentally different functionality between dielectric and gold-coated tips has been clearly identified experimentally by checking against the solutions of Maxwell equations. It turns out that dielectric tips detect a signal proportional to the optical electric field intensity, whereas gold-coated tips detect a signal proportional to the optical magnetic field intensity. The possible implications of this surprising phenomenon are discussed.

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.

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.

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.

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.

Photon scanning-tunneling microscopy of unstained Mammalian cells and chromosomes.

The photon scanning-tunneling microscope (PSTM) yields optical topographical images of samples that are thin or that are transparent at the wavelength used. A range of sample sizes can be imaged extending to well below the diffraction limit for sufficiently flat samples. But samples of the order of several to many micrometers in size can be analyzed with less-refined resolution if total internal reflection can be made to occur in the sample. We used the PSTM to examine the optical topography of mouse and human cells and of chromosomes that are unstained. Our objectives were to demonstrate the images as an alternative to conventional microscopy and to provide a sample-preparation methodology that will later permit localized, simultaneous fluorescence or absorption spectroscopy with the signals collected by the probe tip. Furthermore, the PSTM's ability to produce optical profiles in air and in water was tested to establish the basis for future investigation of possible abnormalities in the chromosomes. That is, we considered both physical and biological objectives. To this end we utilized the 442-nm line of a He-Cd laser as well as the 633-nm line from a He-Ne laser, the resulting image quality being tested partly to ascertain the increased effects of scattering at the smaller wavelength. It is shown that adequate resolution and signal-to-noise ratio can be obtained with the shorter wavelength even in the presence of intensity fluctuations from the laser, thus showing that fluorescence and absorption studies can be expected to be practicable.

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy.

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to be a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.

Imaging of the cytoplasmic leaflet of the plasma membrane by atomic force microscopy.

The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on glass coverslips was imaged by atomic force microscopy (AFM) in air and under aqueous medium, in "contact" mode. Micrometer range scans on air-dried samples revealed a heterogeneous structure with some filaments, likely corresponding to actin filaments that abut the inner leaflet of the membrane, and a few semi-organized lattice structures that might correspond to clathrin lattices. Experiments in phosphate-buffered saline confirmed the heterogeneity of the inner membrane surface with the presence of large (> 100 nm) globular structures emerging from the surface. Using sub-micrometer scan ranges, protruding particles, that occupy most of the membrane surface, were imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, high magnification images of the inner membrane surface were heterogeneous with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proximity.

Imaging of the membrane surface of MDCK cells by atomic force microscopy.

The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions, including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.

Effect of coherence of the source on the images obtained with a photon scanning tunneling microscope.

The photon scanning tunneling microscope is based on the frustration of a total internal reflection beam by the end of an optical fiber. This microscope has been used to obtain topographic information generally on smooth samples. We compare images obtained with different sources, such as He-Ne and He-Cd lasers and white-light sources, and show the role of the coherence of the source on the formation of the images.

Reflection scanning microscopy.

To image nontransparent samples we have utilized a special type of scanning-probe microscope that is referred to here as a reflection scanning microscope. The reflection scanning microscope provides a method for producing a scanned point light source as well as a system for collecting the light that is reflected by the sample. The system, which uses an optical fiber coupler, is easily installed on an existing photon scanning tunneling microscope. A calculation of the coupling coefficient between the natural propagation mode of the optical fiber and the light that is reflected by the sample is presented along with a comparison between calculated and measured values of the intensity of the light that is detected. Several images of different samples are presented that show the actual resolution of the microscope.

Images of 16S ribosomal RNA by scanning tunnelling microscopy.

We report the use of scanning tunnelling microscopy (STM) to study surface topographies of complex nucleic acid structures. From low-resolution STM images of uncoated 16S ribosomal RNA, we demonstrate the possibility of determining several objective parameters (molecular mass and radius of gyration) in order to characterize and identify the molecules observed. These parameters were compared with values obtained by other physical methods and the radius of gyration was found to be the most reliable. At high resolution, it was possible to measure the main dimensions of selected V-form particles more precisely than with electron microscopy. Images of the more compact form have been also obtained that show different domains in the macromolecular structure.

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.

Determination of the refractive index and thickness of a thin film embedded in a given stratified medium.

A thin film deposited on the plane face of a glass substrate or a thin film embedded in a known stratified medium can be characterized by nondestructive optical measurements: transmittance and reflectance for several angles of incidence with s and p polarization. We present here a graphic method for determining the complex refractive index and thickness for thin film optical characterization. Light entering into the medium through a prism or half-cylinder [attenuated total reflection (ATR)] extends the usable angles. Applications are made to very thin metallic films and dielectric films (SiO(x)).