Self-assembly of molecules on surfaces is fundamentally a process whereby individual molecules, separated in the gas or liquid phase, fonn ordered arrays on a surface. The driving forces for these self-assembly processes are the attractive and repulsive interactions among the molecules themselves and between the molecular assemblies and the surface. Since physisorption causes minimal perturbation of chemical bonds, studies of physisorbed species provide an extremely convenient method for investigating issues of molecular structure, conformation, orientation and dynamics of molecules in their “natural” state. The two dimensional nature of the interface introduces constraints that can be used advantageously to separate and identify novel structures.

Control and manipulation of self assembly and molecular conformation is one of the key issues often cited in efforts to develop nanotechnology initiatives for the next generation of electrical and mechanical devices. The development of molecular electronic devices based solely on organic materials will also require careful control and manipulation of molecular assemblies as well as the conformations and orientations of the molecules and functional groups within these arrays.

Numerous scanning tunneling microscopy (STM) studies of adsorbates at 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 of 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 the 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 previous study in our laboratory of a homologous series of para-halogenated phenyloctadecyl ethers (X-POEs, where X = H, CI, Br, I), physisorbed onto highly oriented pyrolytic graphite (HOPG), revealed a bias-dependent contrast in STM images resembling calculated (HyperChem) electron density contours of bonding molecular orbitals.

Proximal Probe Microscopes are emerging as powerful tools for facilitating nanotechnology and nanoscience innovations. Probe microscopes operate by moving a nanoscopic probe across a surface in a raster motion. A surface topographic image is generated by displaying on a computer screen the three-dimensional motion of the probe, and hence a three-dimensional rendering of the surface. in addition to providing a standard magnified view of a surface associated with traditional microscopes, proximal probe microscopes provide quantitative height information. The primary factor establishing resolution in a probe system is the diameter of the probe. with recent advances in probe microscope techniques it is possible to measure more than three-dimensional surface profiles. Examples include measuring surface physical and chemical properties, manipulating objects on a surface, and altering surface properties.

In an atomic force microscope the probe is placed in the vicinity of a surface, and various types mechanical properties may be measured. For example, mechanical properties such a surface hardness and elasticity are measured by monitoring the mechanical interactions between the probe and a sample’s surface. in one such method, the probe is vibrated above the surface and the phase and amplitude of the probe’s vibration depends on the mechanical properties of the surface; Figure 1 illustrates this technique.

We reported recently the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer (CT) were demonstrated. Significant differences were detected in the redox responses given by normal human breast epithelial cells (MCF-10A) and metastatic breast cancer cells (MDA-MB-231). Here, we demonstrate that the intracellular redox potentials and concentrations of redox components in the cell can be evaluated noninvasively by the SECM. Such measurements provide mechanistic explanation of the observed differences in cell redox activities.

In SECM, a microscopic electrochemical sensor, usually called the tip, is scanned over the surface of a sample, and topographic images and maps of chemical reactivity across the surface are obtained. The schematic diagram of such measurements is shown in Figure 1. The oxidized (or reduced) form of redox species initially present in solution reacts at the surface of the ultramicroelectrode tip.

The utility of the atomic force microscope (AFM) for measuring forces is now well established. The AFM is capable of measuring forces from tens of piconewtons to tens of nanonewtons. Systems ranging from the adhesion of clean or functionalized tips interacting with different surface, to singlemolecule measurements of the force required to rupture protein-ligand interactions, to the forced unfolding of single protein molecules have been studied. The adhesion of a clean silicon nitride tip to a clean glass surface is one of the simplest systems to study and was the first system to demonstrate quantized adhesion. AFM cantilevers will adhere to glass surfaces in the presence of water and the force required to separate the surfaces is on the order of 1-6 nN at low pH. The adhesion mechanism is believed to be due to hydrogen bonds formed between the silanol groups present on both the glass and AFM tip. Deuterium oxide (D2O) has physical properties significantly different from that of H2O; the density, melting temperature and heat capacity are all greater than that of water. These and the differences between H2O and D2O can be attributed to the lower vibrational frequency of the OD bond and the fact that O-D…O bonds are about lkJ/mol stronger than O-H…O bonds.

Atomic force microscopy (AFM) allows the surfaces of native biological materials to be imaged in aqueous solution with submolecular resolution. The ability to perform AFM imaging in aqueous and physiological environment has made it possible to monitor important biological processes in real time at high resolution. Currently, there is a great deal of interest in AFM studies of the structure and property of lipid bilayer membranes and protein interactions with lipid bilayer membranes. Lipid bilayer membranes in biological cells form a permeability barrier, which controls the flow of ions, water, and other molecules between biological cells and their environments, whereas membrane-bound and/or membrane-associated proteins are responsible for most of the dynamic functions carried out by the membrane. However, real-time AFM monitoring of dynamic biological processes has been challenged by the limited temporal resolution of AFM, potential physical damage to soft biological samples, and intrinsic complexity of biological processes. There are few successful examples of AFM real-time studies of dynamic biological events, particularly in the aspect of protein interactions with lipid bilayer membranes.

We have attempted to use atomic force microscopy to study interactions between a particular protein, saposin C, and phospholipid bilayer membranes in real time. Saposin C (Sap C), a small glycoprotein, is an essential co-factor for the hydrolysis of glucosylceramide by glucosylceramidase in lysosomes, and a deficiency of Sap C leads to a variant form of Gauchers’ diseases. Supported planar phospholipid bilayer membranes were used in the study.

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.

We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

Recent work has demonstrated the ability to measure forces between and within single molecules using cantilever-based instruments. Force measurements on molecules can provide structural and dynamic information not available with other techniques. Figure 1 shows an example force-distance curve made of lambda digest DNA being stretched by a cantilever, showing the characteristic plateau at 65 pN as the DNA goes through the B-S transition. Implicit in these measurements is a need for carefully quantifying the forces the cantilever is exerting on the single molecule. This requires a low noise system that provides accurate measurements of the cantilever spring constant, cantilever bending, and the relative cantilever-sample separation. We have developed an instrument with several improvements that allows these measurements to be made quantitatively. One component is an improved optical lever that provides an extremely sensitive measurement of the cantilever deflection. A separate sensor measures the position of the moving base of the cantilever and allows thermal drift, piezo scanner hysteresis, and creep effects to be corrected.

In force measurements with micro-machined cantilevers the application of Hooke's law (F = −kx) requires two quantities: x, the distance the flexing cantilever moves and the “spring constant” k. The optical lever is the most commonly used method for measuring the deflection of a cantilever.

The scanning Kelvin probe force microscope (SKPM) is a member of the scanning probe microscope (SPM) family, and was derived from the non-contact atomic force microscope (NCAFM) technique. The contact potential difference (CPD) originating from the work function difference between the tip and sample surfaces can be measured using SKPM with simultaneous observation of the topography image. Using SKPM the surface of semiconductor device has been observed to measure the dopant concentration as the CPD difference in two dimensions. Most of the SKPM measurements are acquired in the atmosphere. The lateral resolution is achieved less than 100 nm.

On the other hand, Scanning capacitance microscope (SCM) of contact mode is now most popular method to measure the dopant concentration in semiconductor device. However, the lateral resolution of SCM is limited, because of the fact that the contact mode cannot achieve a true atomic resolution, the fact that the capacitance is measured through the insulating layer on surface, and the fact that the tip and sample is damaged by the high electric field to be applied between the tip and sample. So SCM never achieves the atomic level resolution.

In recent years, the magnetic force microscope (MFM) has been used not only for the evaluation of magnetic media but also for the measurement of magnetic characteristics of quantum dots as well as in various fields, because the MFM can visualize magnetic field on the sample surface with the high resolution. The MFM observation in ultra-high vacuum (UHV) requires to reduce the adsorption layer on the sample surface for high resolution observation. The slope detection mode using the variation of vibrating cantilever is basically unstable in UHV due to the increase in the Q factor of the cantilever. Accordingly it has been used for the MFM observation in air. On the other hand, the FM detection technique offers a very high sensitivity because of the high Q factor.

This repot describes the MFM technique to take a magnetic image steadily in UHV. We observed the surface of a magnetic material with MFM technique in UHV, and compared the slope detection technique, the phase detection technique using phase variation of cantilever vibration, and the FM detection technique.

To date it has been possible to solve the structure of several membrane proteins to atomic resolution with cryo-electron crystallography (1-4). in all studies two-dimensional crystals were required as well as special provisions on the transmission microscope. in all cases a field emission gun provided the necessary coherence and liquid-Helium cooling the specimen protection (4,5).

Although repeatedly studies have been done on the cryo-protection factor of beam sensitive specimen at liquid-Nitrogen and liquid-Helium temperatures (6-10), the data available are still too limited for a clear demonstration of the superiority of liquid-Helium. Especially investigations are needed with individual particles embedded in amorphous ice (11).

To meet the increasing demand for liquid-Helium cooling, FEI developed the Tecnai F30 Helium. The challenge was to provide an instrument with a very stable cryo-stage, which can work at liquid- Nitrogen as well as liquid-Helium temperature.

Current trends seek to make TEM operation more routine and productive. One of these follows the lead made in scanning electron microscopy in which the spatial distribution of crystallographic data over a selected area of a sample surface is determined by automated recognition of Backscatter Kikuchi patterns (EBSD), Adams et al . This method has been extended to the TEM in a novel manner, Wright et al . Dark field images are collected sequentially from the area of interest for all possible incident beam directions over a solid cone of semi-apex angle up to 4 degrees, Figure 1. The images are recorded directly into a computer. Off line automated analysis follows in which all dark field images are searched to determine the beam conditions under which each pixel in the image brightens, i.e. when the corresponding point in the sample came into a diffraction condition. The measurement of three such non-coplanar conditions for a pixel leads to the determination of the corresponding crystallographic orientation. The accuracy of orientation measurement has been improved to 1 degree by the fitting of the determined diffraction vectors to the surface of the Ewald sphere. The analysis allows grain maps to be reconstructed from which grain size distribution can be automatically determined. Figure 2.

The movement towards a single platform concept for the operation of multi-functional analytical instruments has progressed over the past 10 years largely due to the availability of powerful computer technology combined with the need of ease of use to increase productivity. Other factors include the need to share data obtained from these increasingly complex instruments, and to transport these data in an easily managed format and the possibility of storing large amounts of data generated from projects.

Initial efforts in this field were generated by the need for computer control of complex instrumentation such as TEMs, EPMAs and dual beam instruments. Microprocessors enabled easy alignment, storage of stage parameters and digital image acquisition. with the boom in the semiconductor industry it was necessary to design tools that could be operated by individuals who did not possess the same amount of knowledge as the scientists and technologists of the previous generation. Early attempts at integration of techniques on a single platform were restricted, largely due to limitations in the capacity of the computers employed in controlling these tasks. Indeed, in some cases this actually increased the complexity of some instruments due to a profusion of microprocessors controlling the various analytical components of the larger analytical machines.

The resolution of state-of-the-art low-voltage scanning electron microscopes (LV SEM), which is currently limited by the chromatic and spherical aberrations of the objective lens, can be improved by incorporating an aberration correcting device. At present four different concepts are discussed in literature: Zach and Haider demonstrated that a quadrupole/octupole corrector can correct both chromatic and spherical aberration. Rose proposed a Wien filter for chromatic aberration correction, which has relaxed stability requirements. Recently, we reported a simplified version of this corrector and showed that a spherical aberration corrector can be integrated in a Wien filter. Henstra and co-workers suggested a purely electrostatic corrector that can correct both chromatic and spherical aberration.

For all these concepts problems may arise when the lens-to-sample (working) distance for an aligned corrector is to be changed. in general, the corrector settings depend on the ratio Cc/f2, where Cc and f denote the coefficient of the chromatic aberration and the focal length of the objective lens, respectively. When the working distance is changed, this ratio is no longer perfectly matched to the corrector settings. The tedious realignments and readjustments, which then seem necessary, can be avoided by using a doublet objective lens as illustrated schematically in Figure 1.

Past FE-SEM could obtain a high resolution image, however its probe current was not sufficiently strong enough for analytical purpose.

We have developed a multi-purpose thermal field emission scanning electron microscope (JSM- 6500F), in which a new designed “In-Lens Thermal FEG” is installed.

Fig. 1 shows a cross section images of the In-Lens Thermal FEG, comparing with the past FEG. The In-Lens Thermal FEG consists of the thermal FEG and the 1st condenser lens. The emitter is located in the magnetic field produced by the 1st condenser lens, so that electrons emitted from the emitter are condensed effectively to produced a high probe current. The maximum probe current of 200 nA is attainable at the accelerating voltage of 15 kV, ten times larger than the maximum probe current of ordinary FE-SEMs. Therefore the WDS analysis can be performed by this newly FE-SEM.

The “aperture angle control lens” has been installed in the electron optics system, for improving the resolution at a large probe current. The resolution of 3.0nm at the analytical condition (at probe current 5nA, accelerating voltage 15kV, WD 10mm: fig.2).

The ultimate resolution of the microscope is 1.5nm at 15kV and 5nm at lkV. The objective lens is not an immersion type and does not leak magnetic fields on the specimen surface, therefore this equipment was suitable for observing or analyzing magnetic materials, and also suitable for the EBSD analysis. Fig.3 shows an example of the EBSD analysis.

Electron beam - specimen interactions and SEM signals have been well understood and documented for many years. These interactions result in a variety of electron signals including the most common, secondary and backscattered electron. Each electron signal produces unique characteristic information about the sample surface, subsurface, and elemental composition. Important information can be gained by controlling and filtering electron signals collected by the electron detector system.

The S-4700 Cold Field Emission SEM incorporates a set of electrodes and plates positioned in the objective lens upper pole piece in close proximity to the upper secondary detector (figure 1). When a positive voltage is applied to the electrode plates, a high yield of secondary and backscattered electrons spiral up the column of the objective lens. The backscattered electrons are filtered with the ExB producing a SE rich signal. The information from this type of signal provides absolute detail from the sample surface, but can be prone to charging with some highly nonconductive samples. Figure 2a shows the effect of charging while observing uncoated Teflon ™. The image becomes distorted with bright intermittent horizontal lines. Surface detail is enhanced due to the high contribution of SEs from the sample surface.

When the electrode voltage is set negative, through the instrument GUI, the low energy secondary electrons are repelled providing a signal rich in backscattered electrons. The information from this type of signal provides compositional information and inherently reduces charging. The uncoated Teflon ™ sample in figure 2b shows all charging affects have been eliminated.

To examine non conductive samples in their natural state (i.e. without significant sample preparation) at high resolution in the SEM the technique of low voltage field emission scanning electron microscopy (LVFESEM) is used. Due to the limitation in accelerating voltage (U<1kV) this technique is limited in respect of chemical analysis. Furthermore it is not possible to examine humid and outgassing samples in high vacuum. in recent years the application of variable pressure scanning electron microscopes (VPSEM) became an important technique in materials science as well as in life science. Due to the capability of maintaining a high chamber pressure humid, outgassing and non-conductive samples, can be examined in their natural state without significant sample modification or preparation. Especially compound materials with different electron yields can be imaged without any charging effects (Fig. 2), [2]. This paper describes a high resolution field emission electron microscope, that combines low voltage and variable pressure capabilities.

The high pressure capabilities of the instrument are realized by eliminating the high vacuum requirements of SEM in the microscope chamber. This is done by separating the vacuum environment in the chamber from the ultra high vacuum environment in the gun area.

The use of variable pressure SEMs (VP-SEMs) is increasing in various fields of science and industry, allowing microscopy in a variable pressure environment of 1 ∼ 270 Pa utilizing backscattered electrons for imaging. The VP-SEM allows microscopy of insulated samples without the need for sample preparation. Charging artifacts can be minimized as well. When the VP-SEM is operated with a cooling stage, which allows cooling of samples at −20° and above, vaporization of water from samples is reduced. This permits microscopy of wet samples at close to the natural state for extended periods of time.

Poor S/N ratio and deterioration of resolution, both of which are due to collisions among residual gas molecules and primary/backscattered electrons, have limited the performance of VP-SEMs. For resolving these limitations, we have completed the development of a new field emission VP-SEM which operates with a stable Schottky field emission source, a new environmental secondary electron detector (ESED), and a multi-stage differential pumping system. Fig. 1 shows a sectional view of the column with the differential pumping system. This design allows stable gun vacuum conditions with variable specimen chamber pressure 10 through 3,000 Pa, permitting a pressure difference from the gun by 1011 Pa without problems.

RÖNTEC’s UHV Dewar detectors have established new standards for high resolution, lowmaintenance, low operating cost, and reliability in Si(Li) X-ray detectors. Now, the recently introduced XFlash® series X-ray detectors are enabling new methodologies for microanalysis and element imaging in the SEM. These detectors are compact, liquid-nitrogen-free semiconductor Xray detectors that are based on Silicon Drift Diode (SDD) technology. XFlash detectors produce extraordinarily high count rates with excellent energy resolution and have introduced ultra-fast microanalysis and element mapping to the SEM world. The addition of color to SEM images enables easy visualization of element distributions and allows the microstructural features and compositional variations of different materials to be more readily identified. Persons unfamiliar with electron microscopy can more readily interpret color images than black and white or gray scale images. This new technology has great potential to revolutionize electron microscopy.

RÖNTEC’s UHV Dewar Detector offers the highest long-term stability and best energy resolution ever specified for a commercial Si(Li) detector (less than 129 eV). The UHV design leads to small size and weight (for reduced column loading) along with extremely low nitrogen consumption and low susceptibility to microphonics. The UHV detector never ices up and thus never requires defrosting or warm-ups. It is available with a variety of entrance windows for light element analysis.

The complete and accurate identification of all of the phases present in a sample is the ultimate goal of any materials investigator. The identification should include compositional as well as crystalline measurements for unique characterization. in an SEM, the composition of individual regions can be routinely determined via EDS or WDS analysis. The main limitation is the need to visually identify the individual regions from the images alone. A method to limit this restriction is to collect x-ray maps of the field-of-view. This technique has the benefit of providing the spatial distribution of the elemental species, but requires knowledge of all of the elements of interest before the start of the analysis and composition quantification cannot be performed with data only from the maps. Another disadvantage is the possibility that materials may have similar compositional analyses but may be different phases only manifested by crystallographic means.

A better method of complete phase identification involves the collection and analysis of spectral images and the subsequent crystallographic phase identification via EBSD. Spectral imaging consists of the collection of complete EDS spectra at each location within a field-of-view. From this 3-D data set, a series of elemental maps may be extracted for visualizing the spatial distribution of any elements of interest. This data set is especially useful because elements, and even characteristic x-ray lines, can be fully analyzed after the data collection with total flexibility.