Scanning tunneling microscope combined with scanning electron microscope

A scanning tunneling microscope (STM) has been installed in a usual scanning electron microscope (SEM) with a vacuum of 10⁻⁶ Torr. The STM image is displayed on the cathode ray tube of the SEM, 512 × 512 pixels, with a scanning rate of 80 s/picture. The spatial resolution of the STM is about 1 Å, while that of the SEM is several tens of ångströms. The combined scanning microscope covers a wide magnification range from 10 to 10⁷, where STM covers the high magnification region from 10⁵ to 10⁷.     

Contrast in high-resolution scanning electron microscope images

Current scanning electron microscopes, equipped with field emission guns and high-performance immersion lenses, can achieve spatial resolutions of the order of 1 nm in both secondary and backscattered imaging modes over a wide range of operating energies. The generation and interpretation of images with nanometre-scale resolution relies on a detailed knowledge, and application, of electron-solid interactions. This paper develops the practical steps required to produce a high-resolution image, and discusses the principles which govern image interpretation. Attention is focused primarily on materials which are low in atomic number and density, such as biological tissue, but the results apply after appropriate scaling of the physical parameters to most other materials.     

Surface investigations with a combined scanning electron–scanning tunneling microscope

We have combined a scanning tunneling microscope (STM) with a scanning electron microscope (SEM) for surface investigations of atomically flat surfaces, ultrathin adsorbate films, and material surfaces. The mechanical stability of the hybrid instrument allows high-resolution SEM of samples mounted on the STM stage and atomic resolution with the STM. Experimental results of combined SEM/STM investigations on textured material surfaces, submicron structures, and atomically flat conducting surfaces are presented. An example is given for surface machining with the STM under SEM control.     

A case of Leukonychia with scanning electron microscope observation

Leukonychia is a medical term for white discoloration appearing on nails. The pathophysiologic mechanisms that cause white discoloration are not entirely clear. We processed a case of leukonychia with scanning electron microscope observation and found many crispy, obviously dissociated "layers" in the lower part of the white nail plate. The dissociated "layers" were composed of thick, loose, coarse keratin bundles intertwined with each other. We believe the dissociated "layers" are related to the clinically noted white discoloration appearing on the nails.     

Probe design optimization for a high-resolution scattering-type scanning near-field optical microscope

The scattering-type scanning near-field optical microscope (SNOM) has a probe with a sharp tip for use in high resolution imaging. As sharp a tip as possible is generally considered ideal for the observations, but actually, a sharp tip does not always provide a high resolution SNOM image. We numerically examined the scattering property of the SNOM probe by the three dimensional finite difference time domain method. In this paper, we show the criterion for the ideal scattering probe which satisfies the simple relation between radius and taper angle of the tip.     

The conversion of a field-emission scanning electron microscope to a high-resolution,high-performance scanning transmission electron microscope,while maintaining original functions

Recently, the reliability of field-emission electron guns has increased. In addition, the cost of computer systems for on-line processing has dropped. Hence, we should now consider the use of scanning transmission electron microscopy (STEM) for routine work, especially, in the field of biology where one may expect to utilize digital image processing techniques. An STEM has been constructed, without disturbing the original functions, by converting a commercial scanning electron microscope equipped with a fieldemission gun. The STEM is generally operated at accelerating voltage 30 kV, focal length 7.5 mm, and beam current 1?2 × 10^?10 A. Several improvements have been incorporated for removing the effects of vibration, contamination, and stray magnetic fields. Also, an adjustable detector aperture was utilized. The modified instrument was connected to an on-line digital image processing system for utilizing the information obtained from STEM images. The advantages of the modified system were studied from various viewpoints.     

A combined scanning tunnelling and field ion microscope

The construction of a combined scanning tunnelling and field ion microscope allowing the in situ preparation and analysis of the tunnelling tip is described. The apparatus is based on the Besocke type STM and includes a fast, automated sample approach, a sample transfer manipulator and a tip-cooling mechanism for the FIM operation mode. The design is simple and needs a mechanical feedthrough only for sample transfer. A method has been developed to produce sharp tungsten tips with small clusters on the apex plane. Test-run results of FIM and STM operation modes are discussed.     

A simple cryo-holder facilitates specimen observation under a conventional scanning electron microscope

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories.     

Splitting wood specimens for observation in the scanning electron microscope

This report deals with the principles of splitting wood specimens for observation in the scanning electron microscope and gives some examples in which the splitting is a crucial step in specimen preparation.     

Cutting wood specimens for observation in the scanning electron microscope

This report deals with the cutting of wood specimens for observation in the scanning electron microscope. Several cutting devices and types of knives are described and critically evaluated.     

Design of a scanning probe microscope with advanced sample treatment capabilities: An atomic force microscope combined with a miniaturized inductively coupled plasma source

We describe the design and performance of an atomic force microscope (AFM) combined with a miniaturized inductively coupled plasma source working at a radio frequency of 27.12 MHz. State-of-the-art scanning probe microscopes (SPMs) have limited in situ sample treatment capabilities. Aggressive treatments such as plasma etching or harsh treatments such as etching in aggressive liquids typically require the removal of the sample from the microscope. Consequently, time consuming procedures are required if the same sample spot has to be imaged after successive processing steps. We have developed a first prototype of a SPM which features a quasi in situ sample treatment using a modified commercial atomic force microscope. A sample holder is positioned in a special reactor chamber; the AFM tip can be retracted by several millimeters so that the chamber can be closed for a treatment procedure. Most importantly, after the treatment, the tip is moved back to the sample with a lateral drift per process step in the 20 nm regime. The performance of the prototype is characterized by consecutive plasma etching of a nanostructured polymer film.     

Real time observation of viscoelastic creep of a polymer coating by scanning probe microscope

We have developed a new technique of using the scanning probe microscope to measure the marring resistance that is a very desirable characteristic of clearcoats used in the automotive industry. With its high resolution, SPM can also be used to investigate the marring mechanism, and provide useful information in development of highly mar resistant coatings. In this paper, we will report a real time observation of viscoelastic creep which can result in a partial or complete healing of a marred surface. Based on the experimental data, a preliminary analysis of the recovery rate, recoverable deformation, and permanent deformation is presented. This revised version was published online in June 2006 with corrections to the Cover Date.     

Imaging of various surface properties of fluorescently labelled phospholipid Langmuir-Blodgett films with a combined scanning probe microscope

We present results of phase separation of a single-component system of 1,2-dihexadecanoyl-sn-glycero-3-phospho-[N-(4-nitrobenz)-2-oxa-1,3-diazolyl]ethanolamine in which a liquid-condensed (LC) phase co-exists with a liquid-expanded (LE) phase. Domain formation in the co-existence region was studied using a newly developed combined scanning near-field optical microscope-atomic force microscope (SNOM-AFM). We demonstrate for the first time that the topographic, friction, fluorescence and surface potential distributions for a phase-separated single-component Langmuir-Blodgett film between the LE and LC phases can be simultaneously observed using the SNOM-AFM with a thin-step etched optical fibre probe.     

Biological structures imaged in a hybrid scanning transmission electron microscope and scanning tunneling microscope

A hybrid scanning transmission electron microscope (STEM) and scanning tunneling microscope (STM) is described which allows simultaneous imaging of biological structures adsorbed to electron-transparent specimen supports in both modes of scanning microscopy, as demonstrated on uncoated phage T4 polyheads. We further discuss the reproducibility and validity of height data obtained from STM topographs of biomacromolecules and present raw data from topographs of freeze-dried, metal-coated nuclear envelopes from Xenopus laevis oocytes.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to >1 kHz for line scanning and curve scanning, and >100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to > 1 kHz for line scanning and curve scanning, and > 100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

Imaging 'intact' myofibrils with a near-field scanning optical microscope

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro , naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.     

Design and performance of a high-resolution photoelectron microscope

The design of a high-resolution photoelectron microscope (photoelectron emission microscope) is described. It is an oil-free ultrahigh-vacuum instrument utilizing electrostatic electron optics. New designs are presented for a specimen translator, cathode stage, aperture stop control, electrostatic hexapole stigmator, beam shutter, and camera system. These components could also be used in a low-energy electron microscope (LEEM). The theoretical resolution of this instrument is 5 nm for UV illumination near the photoemission threshold. The photoelectron microscope is now in operation at the University of Oregon, and it is achieving results within a factor of two of this design limit.     

Specialized scanning ion-conductance microscope for imaging of living cells

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.