STM imaging of the complete bacterial cell sheath of Methanospirillum hungatei

Using a large range scanning tunnelling microscope (STM) we have obtained images of the complete outer cell wall of the archaebacterium Methanospirillum hungatei, deposited on a graphite substrate and over-coated with an Au or Pt evaporated film. The width of the collapsed cylindrical sheath structure was found to be 5000 Å ± ***10%, which agrees closely with the value from previously published electron microscope (EM) studies. The double thickness of the collapsed sheath was found to be 160 Å ± 10%, which is about 20% smaller than that from the EM results. Higher resolution STM images taken on top of the collapsed sheaths show corrugations running perpendicular to the cylinder axis and having widths which are multiples of ～ 30 Å, the minimum period expected from EM studies. The height of the corrugations have a minimum value of about 4 Å. The expected 2-D crystalline structure was not seen in the STM images.     

STM measurements on graphite using correlation averaging of the data

Images of the (0001) surface of highly oriented pyrolytic graphite observed using scanning tunnelling microscopy, in both constant distance and current imaging modes, show a strong asymmetry of the unit cell. This asymmetry depends on the tunnelling current and voltage. We have also found clear evidence that multiple-tip tunnelling occurs. To improve the resolution of the imaged unit cells, a method of correlation averaging is introduced.     

The effect of a vacancy on the STM image of graphite

Theoretical studies have been done on graphite with a vacancy using the extended Huckel tight-binding (EHTB) method. The results of the singly- and doubly-layered samples show that there is an unexpected distance dependence of the charge density directly over the vacancy. Furthermore, the vacancy acts as a perturbation to distinguish two sublattices which exist in pristine graphite and it creates a charge enhancement in the atoms directly surrounding it.     

STM and AFM investigations of high-Tc superconductors

We have studied the (001) surface of single crystal YBa₂Cu₃O_7-x high-T_c superconductors using scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) at room temperature at ambient pressure. Both methods show flat terraces with steps which are multiples of the c-axis lattice constant (of 1·17 nm) high. Our results show that the bulk crystal structure extends to the surface and that the crystals were formed by island growth. Only occasionally tunnelling was possible with sample bias voltages below +1·0 V. We interpret the observed voltage dependence and the difficulty to get good STM images to be due to the presence of a less-conducting surface layer. Auger spectroscopy indicates that carbon is present at the surface, which is probably related to a contamination layer.     

Observation of rhombic facets on platinum wire after heating in air using scanning tunnelling microscopy

Evidence is provided for the formation of surface faceting due to heating in air at 1160 K. For the first time diamond-shaped facets with angles of 60 and 120° have been observed on the surface of a sample of ultrapure platinum wire in air using scanning tunnelling microscopy. The maximum extension of the facets is about 1000 Å.     

STM study of the structure of the sulphur (1×2) overlayer on molybdenum (001) in air: ordered domains,phase boundaries and defects

A (001) surface of molybdenum, covered by one monolayer of sulphur was prepared in UHV and characterized by LEED, Auger and XPS. This surface was found to be stable in air for periods of several days. STM images of the surface, obtained in air in the topographic and local barrier height modes, revealed the atomic arrangement of sulphur atoms in domains with 1×2 and 2×1 periodicities. Boundaries between domains, adsorbate and substrate defect structures and crystallites formed during the initial oxidation of the Mo substrate were observed.     

Scanning tunnelling microscopy of 1-D and 2-D charge-density wave systems

We have used a new variable temperature scanning tunnelling microscope (STM) to study quasi-1D and 2-D charge-density wave (CDW) systems. The 1-D systems, typified by NbSe₃ and TaS₃, are of special interest since they exhibit unusual transport phenomena associated with moving CDW above a threshold electric field. In the case of NbSe₃, room temperature STM images show both major and subtle details of the lattice structure. At present, however, images taken below the Peierls transition temperature of T_P=144 K resolve major lattice details but are not sufficiently clear to resolve the CDW. On the other hand, for the fully gapped CDW system orthorhombic-TaS₃, the CDW modulation superimposed on the lattice structure and having the correct period of four times the S-S spacing of 3·3 Å, is observed below T_P=215 K. Above T_P, the main observable feature is the S-S spacing along the chains. STM measurements have also been performed on the 2-D CDW system 1T-TaS₂ in its incommensurate, nearly commensurate, fully commensurate and trigonal phases. For the nearly commensurate phase, STM images show uniform commensurability with a relatively low concentration of small, time-varying discommensurations in contrast to models pradicting a regular domain structure. In the trigonal phase, however, evidence is seen for the striped phase composed of long, nearly parallel discommensurations.     

STM study of the effects of pulsed laser irradiation on semiconductor surfaces

A clean Si(111) flat surface was irradiated by pulsed Nd-YAG laser beams. STM observation of the irradiated surfaces indicates that the laser irradiation generates two kinds of corrugations: rounded hills 100 Å high and 600 Å wide and small spikes 20 Å high and 35 Å wide. The corrugations grew and spread with repeated irradiation and increased laser power. The heating of the irradiated surface at 1200°C for 5 s smoothed the surface atomically, but no ordered structure was observed, possibly due to the covering contaminants released from the surfaces surrounding the specimen by laser irradiation and heating.     

Scanning tunnelling microscopy of charge density waves in 1T-TaS2

Using a variable temperature scanning tunnelling microscope we have observed both the lattice and the triple charge density wave (CDW) in 1T-TaS₂. We obtained images of the nearly-commensurate (NC) phase between 350 and 200 K and the triclinic (T) phase between 220 and 280 K. We analysed the images to determine the local angle of rotation of the CDW relative to the lattice to determine if commensurate domains exist in this material. In the NC phase we do not see the hexagonal discommensurate domains proposed by Nakanishi el al. (1977), but observe a structure which is uniformly incommensurate. The orientation of the CDW relative to the lattice is observed to be locally incommensurate and to vary continuously with temperature as reported by Scruby et al. (1975). However, in the T phase we observe long narrow commensurate domains very similar to those described by Nakanishi and Shiba (1984) for this phase.     

Platinum/iridium/carbon: a high-resolution shadowing material for TEM,STM and SEM of biological macromolecular structures

Thin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze-dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high-resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron-beam-heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre-evaporated. In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength-dispersive X-ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI-layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three-dimensionally stable on the freeze-dried macromolecular samples. By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface-sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze-dried biological structures because of its good electrical conductivity and its direct three-dimensional stability. We compare STM, SEM and TEM images of freeze-dried and Pt/Ir/C-coated polyheads.     

Adsorbate deformation as a contrast mechanism in STM images of biopolymers in an aqueous environment: images of the unstained,hydrated DNA double helix

A stable residual aggregate remains on a submerged gold surface after electrophoretic deposition of DNA. We present scanning tunnelling microscope (STM) images of these aggregates which show many objects with the geometry of DNA, clearly displaying the 3·4 nm helix pitch. These images are quite distinctive, and cannot be generated when the deposition technique is used without DNA in the buffer solution. A characteristic of these images is that the tip is observed to dip down over the DNA molecule at the same time as the apparent barrier height drops by a factor of about four. The tip displacement is accounted for by a model in which contrast is dominated by local fluctuations in the deformability of the adsorbate layer, a quantity deduced from measurements of the apparent barrier heights in air, water, over small molecule adsorbates, and over DNA.     

A role of a tip geometry on STM images

Scanning tunnelling microscopes operable in both ultrahigh vacuum (UHV) and aqueous conditions are described with emphasis on a tip geometry. An atomically resolved STM image was obtained for the Si(111)-Mo√3 surface in UHV condition and changes of a diffraction grating surface in aqueous condition due to an electrochemical reaction were monitored.     

Voltage-dependent imaging of antimony on the GaAs(110) surface

Using a scanning tunnelling microscope, voltage-dependent imaging of antimony on the GaAs(110) surface has been performed. For negative sample voltages, the images reflect surface dangling bonds. Depending on the magnitude of the voltage, either one or both of the Sb atoms in the surface unit cell are seen. At positive voltage the density of surface-states is observed to be greatly reduced compared to negative voltages. The results are analysed within the context of a simple tight-binding model for a one-dimensional biatomic chain.     

Metal-induced reconstructions of the silicon(111) surface

We have used scanning tunnelling microscopy (STM) to study changes in the structure of the Si(111) surface induced by deposition of the group III metals In and Ga. For both metals, several different ordered reconstructions are seen as a function of coverage. The STM images provide new structural information on each of these reconstructions. With In metal deposition, we have seen the surface reconstructions √3×, √3, √31× √31, √7×√3 and 4×1 as the coverage increases. In the case of Ga on Si(111), we have studied structures that exist up to 0·7 ML. At 1/3 ML, there is a √3×√3 structure identical to that of In. Above 0·3 ML there is a different phase that may correspond to the (6·3times6·3) RHEED pattern reported in this coverage range. This surface tends to grow as triangular islands at higher coverages.     

Compensation of the shadowing error in three-dimensional imaging with a multiple detector scanning electron microscope

A method for three‐dimensional quantitative surface characterization for scanning electron microscopy is presented. The method used a quadruple scintillator detector developed by us. A surface reconstruction algorithm was performed by special software, with new algorithms for error compensation. Among these errors, detector shadowing was of particular importance. This was due to the disturbance in integration continuity when one or more detectors was screened from the flow of electrons. Several methods for the reduction of this error have been proposed and tested by us. The methods were based on software processing of complementary information, such as unshadowed detector signals, shadow depth and modified integration schemes.     

The STEM approach to the imaging of surfaces and small particles

The characteristics of scanning transmission electron microsopy, which make it a particularly powerful tool for the study of surfaces and small crystals, include the serial nature of the image signal which makes it amenable to recording and image processing procedures, the possibility of correlating the image data with microdiffraction patterns and microanalysis of chosen small regions and the possibilities for combining several different image signals obtained simultaneously. Images obtained from surfaces using reflected diffraction beams may be very sensitive to variations of surface structure but the resolution, depth of focus and contrast of the images are strongly dependent on the nature of the surface and the aperture sizes used in the microsope. Microdiffraction patterns may be obtained from small surface features. Electron energy loss analyses of the diffracted beams provides sensitive indications on surface electron excitations. Microdiffraction patterns obtained in transmission from regions of 1–5 nm diameter provide valuable information on the structure and defects of small crystals, such as those of platinum and gold which are of interest in relation to the study of catalysts.     

Reflection electron imaging and spectroscopy studies of aluminium metal reduction at α-aluminium oxide (0,1,2) surfaces

Cleaved α-aluminium oxide (0,1,2) surfaces are studied using combined techniques of scanning reflection electron microscopy and reflection electron energy-loss spectroscopy. An α-aluminium oxide (0,1,2) surface can terminate with a layer of either oxygen or Al atoms depending on where the bonds break. Aluminium metal can be reduced only from the domains initially terminated with oxygen by an intensive electron beam. The interpretation of this damage effect is based on the desorption process of oxygen atoms after Auger decay. The resistance to beam damage of the domains initially terminated with Al is found to be due to the protection of the surface adsorbed amorphous oxygen layer.     

A novel scale‐down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate

At the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale‐down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof‐of‐concept study, TEM specimen supporters (10–30 μm thick) with fine controlled open pores (100～600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free‐standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high‐quality micrographs of cell–cell and cell–scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled‐down design was able to improve our mechanistic understanding of tissue formation not only at single‐ and multiple‐cell levels, but also at micro‐ and nanoscales, which could be difficult to obtain using other methods.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.