Self-assembled monolayers (SAM) and multilayers of organic materials have been intensely studied in the past years, due to their numerous potential applications as, for example, lubricants, corrosioninhibitors and/or adhesion-promoters [1,2]. In this talk, we will present results of several studies carried out using Atomic Force Microscopy to investigate octadecylphosphonic acid (OPA) SAM deposited on mica. We have assessed various mechanisms of assembling, disassembling and reassembling the SAM on mica. The SAM deposition method employed in this work was drip coating using an OPA solution. We have used two different solvents, which exhibit very different OPA solubility, in this work: ethanol and trihydrofuran (THF). Regarding the assembling studies, we will show the formation of groups of OPA double-layers as the initial deposition stage (deposition time < 2 seconds) when using ethanol-based solutions. We will also show that annealing such samples at 60°C produces, favorably, OPA quadruple-layers, as shown in figures la and lb, respectively.

Numerous scanning tunneling microscopy (STM) studies of organic adsorbates at the gas/solid and liquid/solid interfaces have been reported. Although early STM experiments of these systems were concerned primarily with visualizing molecules at the atomic level, the focus has shifted to extracting chemical information from STM images, including the identity of atoms or functional groups within an adsorbed molecule. However, STM image interpretation continues to be an immense challenge and one currently debated issue of critical importance is the mechanism(s) by which image contrast reveals atomic and molecular structure. Recently, a combination of electronic and geometric factors was proposed. The electronic factor addresses the coupling between the energy levels of the adsorbate and the Fermi level of the surface whereas the geometric factor is related to the spatial overlap between the STM tip and the functional group.

A study in our laboratory of a homologous series of para-halogenated phenyloctadecyl ethers (XPOEs, where X = H, Cl, Br, I) adsorbed on highly oriented pyrolytic graphite (HOPG) has contributed significantly to the understanding of the image contrast mechanism for molecular adsorbates and of the chemical specificity of STM.

Quantum dots and nanocrystalline films are interesting materials due to their novel properties, not achievable from the bulk materials [1]. New materials fabricated by assembling of quantum dots and nanostructured materials exhibit, for example, high optical nonlinearities. Fractal structures [2] can be formed from quantum-dot aggregates or films. Scanning probe microscopy is the essential technique to characterize these nanometer-scaled materials?

Gold nanocluster colloids are synthesized in the interior of surfactant aggregates known as inverse micelles, without the use of water to solubilize the metal salt [3]. The size of the Au quantum dots is well controlled in the colloid synthesis and selected using chromatography. The Au clusters are then sprayed onto a glass slides to form thin films made of Au quantum dots. We have also used the laser ablation technique [4] to grow Ag nanoparticles, fractal aggregates and thin films. These samples have been studied using atomic force microscopy (AFM) and nearfield scanning optical microscopy.

Scanning Probe Microscopy (SPM) is a superb tool for topographical analysis of samples. However, traditional varieties of SPM such as Atomic Force, Scanning Tunneling and Near-field Scanning Optical Microscopy have limited chemical contrast capability. Recently, several advanced techniques have been reported which provide chemical information in addition to topographical data. All these methods derive advantage from combinations of scanning probe methodologies and some other, chemically sensitive technique. Examples of such approaches are: Near-field Scanning Raman Imaging, Near-field Scanning Infrared Microscopy and mass spectrometric analysis with laser ablation through fiber probes.

In this contribution we report the development of a new method in this family of chemically sensitive scanning probe techniques: Laser Induced Breakdown Spectroscopy with Shear Force Microscopy, LIBS-SFM. Traditional LIBS experiments involve focusing a pulsed laser beam onto the sample and observing optical emission from the plasma formed in the ablation area. The emissions are mostly in the UV/visible range, and the signal is due to electronic transitions in excited atoms and ions in the plasma plume. The spectra are analyzed to identify chemical elements. The spatial resolution of LIBS is limited by the wavelength and beam quality of the laser used for ablation. The experiments may be conducted in vacuum, controlled atmosphere, or ambient air.

The atomic force microscope (AFM) is a powerful tool for probing surface topography, in addition to chemical and mechanical properties of surfaces. To investigate the chemical properties of a sample, force measurements are made between the AFM's “molecular finger,” or probe, and the sample. Force measurements can be made in many environmental conditions, which makes the AFM a versatile technique. However, in ambient conditions most samples are covered by a thin layer of water. When the AFM probe comes into contact with the thin water layer, a capillary bridge forms between the AFM probe and sample. Because of this water layer, it is believed that in ambient conditions a non-surface specific capillary force dominates the adhesive force between the AFM probe and sample. The non-surface specific capillary force should obscure any information about the surface. Currently, we are studying to what extent the capillary forces are non-surface specific and what influence this has on pull-off-force measurements.

In a number of situations and applications, the tribological wear of ceramic and minerallike surfaces is often greatly enhanced in the presence of a corrosive fluid. Examples are the wear of surfaces associated with joints (biomaterials) subject to and the extremely active application to high density circuit production which requires chemical mechanical polishing (CMP). The physics and chemistry of these enhanced wear rates are needed to reduce wear and to improve and extend CMP to new applications. In both situations, surfaces experience simultaneous tribological loading and corrosive chemical exposure, which together produce high wear rates and altered topography. In CMP, the goal is to efficiently achieve very flat surfaces. As feature sizes diminish further, it is conceivable that atomically flat surfaces will be necessary. In almost all tribological situations, it is the interaction of asperities, often micron and sub-micron in dimensions, that control the wear processes due to the high stresses associated with these small structures.

It is important to be able to image biological samples in a manner that is non-invasive and allows the sample to retain its functionality during imaging.

A member of the SPM (scanning probe microscopy) family, SNOM (scanning near-field optical microscopy), has emerged as a technique that allows optical and topographic imaging of biological samples whilst satisfying the above stated criteria. The basic operating principle of SNOM is as follows. Light is coupled down a fibre-optic probe with an output aperture of sub-wavelength dimensions. The probe is then scanned over the sample surface from a distance that is approximately equal to the size of its aperture. By this apparently simple arrangement, the diffraction limit posed by conventional optical microscopy is overcome and simultaneous generation of optical and topographic images of sub-wavelength resolution is made possible. Spatial resolution values of lOOnm in air and 60nm in liquid[1,2] are achievable with SNOM.

Scanning capacitance microscopy (SCM) is currently one of the most promising tools for twodimensional carrier profiling. This technique, based upon atomic force microscope (AFM) operated in the contact mode, uses a conductive probe which is scanned over the semiconductor surface. The conductive probe, oxide on the surface of the semiconductor, and the semiconductor substrate form a metal-oxide-semiconductor (MOS) structure. An a.c. bias is applied to the tip and the capacitance of the MOS structure is monitored. The a.c. bias changes the depletion of carriers in the semiconductor and thus the total capacitance of the structure. The maximum capacitance of the MOS structure is obtained when the semiconductor is in accumulation and the total capacitance of the structure is the capacitance of the semiconductor surface oxide. The minimum capacitance is obtained when the semiconductor region under the tip is in inversion. Since SCM the output signal is proportional to the differential capacitance, to get a high signal we need to maximize the difference between the maximum and minimum in the total MOS capacitance.

In “ac” scanning thermal microscopy, an “active” thermal probe is used also as a heater, so as to inject evanescent temperature waves into a sample and to allow sub-surface imaging of polymers and other materials [1]. The sub-surface detail detected corresponds to variations in heat capacity or thermal conductivity. By suitably choosing the temperature modulation frequency, and hence the penetration depth of the wave, we control the depth of material below the sample surface that is contributing to the image.

Micro-Thermal Analysis [2, 3] builds upon this technique, in order to add spatial resolution to two well-established methods of chemical fingerprinting, DTA and DMA. In both cases, a temperature ramp is used to subject the sample to “events” such as a glass transition or melting. The chief advantages of using the active thermal probe to provide the temperature ramp as well as the modulation, without the use of a heating stage, are: (a) the data are obtained from localised regions chosen from a previously-obtained thermal image, (b) apart from these regions, the rest of the sample is preserved in its original unheated state.

The performance of many industrial polymers is determined by the microscopic morphology of the polymers. For example, surface morphology can influence properties such as adhesion, friction, sealing, blocking, printability, wettability, and haze. Furthermore, bulk morphology often controls mechanical properties such as toughness. strength, wear, and tear resistance. In order to optimize polymer performance, quick reliable methods of determining surface and bulk morphology are essential.

In the past, electron microscopy (in particular TEM) has been the primary method for determining polymer morphology. However, the usefulness of electron microscopy has been limited by the destructive nature of the electron beam, the naturally poor contrast between polymer types, and the difficulty in preparing (staining, etching, cryogenic ultramicrotoming, etc.) high quality specimens.

Recently, the tapping phase-shift mode of atomic force microscopy (TPSAFM) has provided the polymer scientist with a simple, quick, flexible and quantitative method for determining polymer surface and bulk morphology.

Mechanical properties were determined from the organic matrix of a mollusc hard tissue using a nanomechanical testing instrument attached to an atomic force microscope (AFM). Shells of molluscs are two-tier functionally-gradient ceramic/polymer biocomposites used by organisms as impact resistant materials. The inner section of the red abalone (Haliotis refuscens)shell, nacre (mother-ofpearl), has a brick and mortar microarchitecture with pseudo-hexagonal-shaped bricks made of aragonitic (orthorhombic) CaCO3 platelets (0.25 μn thick, and 5 μm edge length) surrounded by a thin (10-25 nm thick) proteinaceous organic matrix (Fig. 1). This higly ordered biocomposite has submicron-layered structure that result in excellent bulk mechanical properties, not achieved by manmade composites with similar phase compositions and, therefore, offers biomimetic lessons for novel materials design. In this model biomimetic composite, mechanical properties of its constituents need to be evaluated in addition to the details of the microstructure. We previously determined nanomechanical properties (hardness and modulus) of individual (twinned, single crystalline) aragonitic platelets.

In industrial applications, such as paints or adhesives, composite latex particles are very common. Most of the properties which are obtained from composite latexes and their films cannot be achieved by physical blending of different polymer components. Therefore it becomes crucial to examine the morphology of composite latexes formed by a multiple-stage polymerization process. The latexes for this study have been examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Various staining methods were used such as ruthenium tetroxide (RuO4), cesium hydroxide (CsOH), and uranyl acetate (UAc) as a negative stain.

The poly(n-butyl acrylate/ poly(methylmethacrylate) (PBA/PMMA) composite latex particles consist of a soft core phase and a hard second phase with varying amounts of acrylic acid in the core, the shell and in both core and shell. The latexes were examined before and after cleaning by a centrifugation technique with 100,000 molecular weight filters.

Confocal microscopy was developed in 1957 by Minski, who was awarded a patent for this work in 1961. Since that time many advances have been in new designs and implementations. There are two basic classes of confocal microscope: the Nipkow-disk-based confocal microscope which allow real-time direct viewing of the sample, and the confocal scanning laser microscopes (CSLM). The CSLM will be focussed on in this presentation.

The CSLM scans a focussed beam over, across or through the sample, collecting the reflected, scattered or emitted light. This light is directed towards an optical spatial filter, which passes light returning from the on-axis focus position, and rejects light that is returning from anywhere else. By detecting the light passing through the spatial filter synchronously with the moving beam and/or sample, an image that has only in-focus information is acquired. Typically a set of images is acquired as the sample is moved through focus: from this “image stack” both an extended-focus image and topographic information can be obtained.

Applications of scanning force microscopy (SFM) in polymer studies have flourished in this decade, reflecting (a) the power of SFM to image both structure and propertiesdown to the nanometer scale, and (b) the low cost and ease of getting useful results in ambient environments. One difficulty in SFM of polymers has been damage incurred by soft materials during the imaging process. The problem was alleviated by the development of special dynamic modes of operation, in which the probe spends little or no time in contact with the polymer surface. Such modes were dubbed “tapping”, “intermittent-contact”, “non-contact”, “near-contact”, etc. As studies proliferated, it became apparent that different researchers were using different terms to refer to the same apparent imaging mechanism, or the same term to refer to different imaging mechanisms. This quandary derived from a poor understanding of exactly how the SFM probe interacts with the sample surface.1-3,5

Fourier Transform Infrared (FTIR) Microspectroscopy is a powerful method to examine and characterize domains down to areas of 10х10 μm2. Spatial concentration maps of chemical species were obtained using apertures to sequentially examine areas on a grid and obtain a map by plotting average spectral absorbance data obtained from each of the areas. New Focal Plane Array (FPA) detectors, composed of a large number (in our case, 64х64) of small detectors arranged in a grid pattern, allow simultaneous collection of infrared radiation at many points from a large spatial region (500х500 μm2 in our case). This allows for a rapid acquisition of chemically specific images from a given area and the examination of several real-time processes. This technique has found a wide variety of applications in multi-phase polymers including polymer laminates, phase separated polymer composites, semi-crystalline polymers and blends, and solvent diffusion into polymers have been the main areas of interest in non-biological IR studies using FPA detection. We present some of the recent work in our group on various multi-phase polymer systems using FPA based infrared spectroscopy.

The diffraction of visible light limits the spatial resolution in conventional optical microscopy to about 200-300 nm. In near-field scanning optical microscopy (NSOM), resolution is improved by bringing the light source, such as the end of an optical fiber, very close to the sample surface. Laser light coupled into the opposite end of the fiber propagates down the fiber core and is emitted from the aperture of the tip. When the sample is in the near-field(roughly within one tip diameter of the end of the tip), the spatial resolution is essentially equal to the diameter of the aperture at the end of the tip and is not determined by diffraction effects. Two-dimensional imaging is accomplished by raster-scanning the sample underneath the fiber tip and collecting transmitted or reflected light at a photodetector.

Atomic force microscopy (AFM) allows the surfaces of native biological macromolecules to be imaged in aqueous solution with submolecular resolution. Short range forces govern the interactions between the AFM tip and the sample and produce the submolecular information of high resolution topographs. In contrast, the long range electrostatic double-layer (EDL) force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area [1]. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteineous substructures. Consequently, the deformation of the native protein is minimized and polypeptide loops of individual proteins were imaged in aqueous solution with a lateral resolution < 5 Åand a vertical resolution < 1 Å (Fig. 1).

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.

Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

DNA in the eukaryotic nucleus is not naked. Instead, it is complexed at frequent intervals with an equal molecular mass of histones to form nucleosomes. In the nucleosome, ∼146 bp of DNA are wrapped around a core histone octamer composed of two copies each of histones H4, H3, H2B, and H2A. The family of linker histones (H1, H5 and H1°) bind to the DNA between successive nucleosomes and help maintain the three-dimensional arrangement of nucleosomes within the chromatin fiber. AFM studies of chromatin fibers with the histones either selectively trypsinized or selectively reconstituted demonstrate a specific role for the H3 N-termini in maintaining fiber structure, in conjunction with the 80 amino acid linker histone globular domain. These AFM results structurally agree with the location of the H3 N-termini in the histone octamer or the nucleosome core particle.

The mechanical Q-factor of an AFM cantilever immersed in fluid is reduced to a small value (ca. 3) owing to viscous damping. Thus, a large driving force is needed to excite the cantilever into bending motion in fluid. There are two common methods for exciting cantilevers for dynamic force microscopy in fluids, illustrated in Figure 1. Fig. la illustrates acoustic excitation in which a piezoelectric transducer displaces the base of the cantilever, causing bending motion of the cantilever when the driving frequency is near to a bending resonance of the cantilever. Fig. lb shows magnetic excitation. In magnetic excitation, a magnetic field is used to cause bending of a magnetic cantilever either through magnetostriction or MXB forces.

Acoustic excitation has the highest amplitude at mechanical resonances of the cantilever housing, with the result that the response is dominated by these sharp features,Fig. 2a. In contrast, the response to magnetic excitation is intrinsic to the cantilever, Fig. 2b. Thus, magnetic excitation permits the cantilever to be driven over a wide range of frequencies. This is important for calibration of the amplitude and for experiments involving time and concentration dependence in tip-sample interactions, e.g., anti-body recognition imaging.