SEM image using secondary electrons (SE) and/or backscattered electrons (BSE) supplies us with elemental and topography information of the sample surface. The SE and BSE characteristics are discussed using the simulated behavior of penetrating electrons in the sample. For the lower incident-energy (≤several hundred electron-volts), the primary electrons more lose straightness of their penetrations and their spatial ranges become to be as shallow as the SE maximum escape depth of about 10nm. This makes both the SE/BSE images more sensitive to the most surface layers and the low-energy BSE nearer to the high-energy SE in their characteristics. Adjustment of the SE/BSE imaging conditions is important to clearly obtain desired information of the sample.
A position of a secondary electron detector or a backscattered electron detector gives large influence on image contrast and illumination effect. Strong illumination effect brought by an out-lens detector used in a conventional SEM gives topographic image contrast with directional shadow. A TTL detector used in an ultrahigh resolution SEM gives weak illumination effect and shadow-less topographic contrast. Morphological information and compositional information can be distinguished by selecting energies of detected electrons at a low accelerating voltage. Stronger illumination effect can be obtained with a lower detector combined with a snorkel objective lens, but an apparent detector position changes according to the change of working distance. Compositional information and topographical information can be separated by calculating output signals obtained from backscattered electron detectors placed symmetrically with respect to the optical axis.
Distribution of crystal grains with a size of a few tens of nanometers was successfully observed in backscattered electron (BSE) images of SEM with a high contrast. An outermost memory layer of DVD-RAM with a thickness of about 20nm was used as a specimen. The observation was made with JSM-7500F, which was capable of impressing bias voltage on the specimen stage to decelerate the incident electrons to lower the landing voltage. A BSE detector was installed in a position of shorter working distance. Voltage ranges covered in the present experiment were from 0.8 to 30kV for the landing voltage and from 0 to 2kV for the bias voltage. The highest contrast was obtained at the landing and the bias voltage of 0.8kV and 0.5kV, respectively.
The image contrast of the scanning electron microscope (SEM) depends on signal generation efficiency of secondary electrons (SE) and the position of the SE detector. The detector of the SEM not only detects secondary electrons but also SE signal generated by backscattered electrons (BSE). It is also difficult to observe the surface of the sample using high accelerating voltage. The realization of high resolution at low accelerating voltage was achieved by a field emission electron gun and a strong excitation type object lens. An SEM equipped with a strong excitation type objective lens includes a control function that can observe an image while adjusting SE and BSE signal with a signal detector. This paper introduces the ExB system within a snorkel type objective lens system with a controllable SE/BSE signal function.
State-of-art SEM allows detection of various SE and BSE signals. These signals can be recorded simultaneously with single scan. As the variety in signal detection increases, the image interpretation becomes more important and difficult. Signal detection in a SEM is complicated; it depends not only on the detector geometry and collection efficiency but also on other factors such as geometry of devices in chamber and the electron optical design. In this report, I will overview the principle of SE and BSE image formation in a SEM with the magnetic/electrostatic compound lens system.
Recent advances in low vacuum scanning electron microscopy (LVSEM) have enabled ultra-high resolution secondary electron (SE) imaging of non-conductors, low voltage scanning transmission electron microscopy (STEM) of hydrated and liquid materials, and SE imaging at gas pressures greater than 1kPa. These developments, driven by novel detection strategies that extend the operating parameter space of conventional LVSEM systems, are discussed and illustrated using state-of-the-art images of material systems such as photolithographic masks, aero gels, nanowires, fake snow and influenza virus.
By using nano-probes attached on a custom made TEM holder with a piezo actuator (TEM-STM holder), narrow current paths composed of several to several tens of nano-particles were selected. The tunnel conductance of the nano-particle system was measured inside a TEM during image observation. The characteristic features of single electron tunneling were recognized at room temperature. The in-situ TEM-STM method is useful for conduction measurements of nano-regions.
Recent studies have revealed frequent occurrence of gap junctions between neurons of particular types. However, for technical reasons it was almost impossible to know the architecture of gap junction-coupled networks. We have developed a new method to visualize three-dimensional organization of gap junction networks by a correlated CLSM-EM. Gap junctions among GABAergic interneurons in the cerebral cortex establish dense and continuous networks that extend laterally in a boundless manner. Structural plan of gap junction networks is implicated for mechanisms of representation of information in the brain.
Quantum dots (Qds) are nanocrystal semiconductor fluorophores consisting of a cadmium selenide core and zinc sulfide or cadmium sulfide shell. They have many advantages over conventional fluorophores including prolonged signal due to photostability, and reinforcement of weak positive reactions. In addition, the confocal laser scanning microscopic (CLSM) dye spectrum analysis system (META, Carl Zeiss, Germany) is utilized. This system ensures optimum specimen illumination and efficient collection of reflected or emitted light and uses an innovative way of separating fluorescent emissions.
In this report, we have demonstrated that multiple Qds signals simultaneous detection from proteins and its transformed factors by CLSM-META. In addition, weak signals produced by conventional immunofluorescence and/or enzyme-labeled antibody methods have been significantly enhanced using Qd labeling, and comparable signals from Qds in the same specimen area have been detected in both transmittance and META modes.
In summary, we believe that Qds represent a breakthrough for the fluorescent antibody method, expanding its already very wide application, and have demonstrated the potential of Qds for the observation of fluorescence microscopy, CLSM, and immunoelectronmicroscopy.
Our results suggest that multiple very weak immunoreactions seen by traditional immunohistochemical techniques can be greatly intensified, a useful feature for widely field.
In the field of life science, optical measurement and imaging technique have recently been key technology to explore the mechanism of biological phenomena, particularly in the monitoring of gene expression using fluorescent or luminescent reporter. In this paper, technologies of super-high sensitive imaging of luminescence and tomographic imaging of fluorescent molecules for biomedical measurement are described.
The ultrahigh voltage electron microscope at Osaka University operates at 3MV, the world-highest accelerating voltage, and it therefore attracts world-wide attention of application researchers in various fields. To meet their requirements, design of a remote operation system for the 3MV UHVEM, which makes it possible to use the EM from remote laboratories in the world, has been studied in our research center. In this paper, contents of the development will be described, with emphasis placed on the following items: (1) necessary conditions for the remote operation, (2) remote operation with a knob box or with a PC installed with a specific software, (3) image transmission with a DV mode by a conventional NTSC or with a HiDVTS mode compatible with the high-vision images, (4) communication link, (5) the way of mutual communication between the laboratories, (6) recording of dynamic images, (7) recording of static images, (8) sharing of recorded images among collaborating laboratories, (9) examples of remote operation and observation, and (10) future prospects.
The flexibility in transmission electron microscopy (TEM), required when converging the beam or switching modes between imaging and diffraction, is provided by a combination of lenses. This paper extends the previous discussion based on geometrical optics to the two-lens system in order to understand the principle of the operation, as well as some practical applications. It also introduces possible consequences of the correction of spherical aberration of the objective lens on the resolution of phase contrast images. That is, it is no longer the contrast-transfer function but the information limit itself that determines the ultimate resolution. It is also pointed out, however, that one needs to select an optimal defocus in order to achieve a better resolution with a reasonable contrast.
The present article reviews the organization of fiber connections of the rat retrosplenial cortex (RS), which lies in the caudal part of the cingulate cortex located in the medial aspect of the cerebral hemisphere. The RS has reciprocal connections with the frontal, parietal, occipital, and retrohippocampal cortices. Most of these connections are topographically organized such that the rostral RS connects with caudal frontal, parietal, and septal retrohippocampal cortices, whereas the caudal RS connects with rostral frontal, occipital, and temporal retrohippocampal cortices. Furthermore, the RS has intrinsic interconnections between subregions of the RS. The RS also connects with subcortical regions such as the striatum, dorsal and ventral thalamus, superior colliculus, midbrain central gray, and pontine nuclei. These corticocortical and subcortical connections of the RS may constitute part of the neural circuitry that underlies spatial memory and navigational functions of the RS.
The electron irradiation effects in carbon nanomaterials are studied by the molecular dynamics simulation including the elastic collision between an incident electron and a carbon atom. The scattering angle of the incident electron is determined using the screened Rutherford cross section. The transferred energy from the electron to the carbon atom and the scattering angle of the carbon atom are obtained by the binary collision theory. The structural changes in the single-walled carbon nanotube, double-walled carbon nanotube, graphite and C60 peapod by the electron irradiation are calculated with the simulation.
In order to analyze the defensive mechanisms against high concentrations of CuSO4 in the copper-tolerant yeast, Cryptococcus liquefaciens (N6 strain), morphology analysis and analytical electron microscopy were conducted on. Copper was detected in strain N6. Copper was distributed in the cytoplasm, rather than in small vacuoles. These results suggest that cytoplasmic and/or some other proteins with a high affinity to heavy metals, may have a role in the defensive mechanisms against high concentrations of CuSO4, as a chelator, in the N6 strain.
The vascular networks in the brain are easily observed by filling vessels with the India ink, acrylic resin, fluorochrome-labeled gelatin and so on. The three dimensional distribution of the vascular networks in the brain can be observed with a confocal laser scanning microscope by filling the vessels with fluorochrome-labeled gelatin. However, it is confined to a part of the brain because of the limitation of the thickness of the specimen. Recently, we have developed and reported a novel method for acquiring serial images suitable for three-dimensional reconstruction of vascular networks in the whole brain of the mouse. In this method, the brain was infused with a White India ink-gelatin solution and embedded in paraffin containing Sudan Black B. The series of serial images were acquired from each sliced surface of the paraffin block without distortion and a problem of the alignment and registration of adjacent images. The three-dimensional image reconstructed from the serial images by the volume-rendering indicated the vascular networks in the whole brain. This volume-rendered image have made it possible to examine the vascular network in the whole brain from any direction, and will be helpful for the investigation of the brain vascular system in various fields.