Yasunori Nawa

Shizuoka University, Sizuoka, Shizuoka, Japan

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Publications (9)12.99 Total impact

  • Yoshimasa Kawata, Yasunori Nawa, Wataru Inami
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    ABSTRACT: We have developed electron beam excitation assisted (EXA) optical microscope[1-3], and demonstrated its resolution higher than 50 nm. In the microscope, a light source in a few nanometers size is excited by focused electron beam in a luminescent film. The microscope makes it possible to observe dynamic behavior of living biological specimens in various surroundings, such as air or liquids. Scan speed of the nanometric light source is faster than that in conventional near-field scanning optical microscopes. The microscope enables to observe optical constants such as absorption, refractive index, polarization, and their dynamic behavior on a nanometric scale. The microscope opens new microscopy applications in nano-technology and nano-science.Figure 1(a) shows schematic diagram of the proposed EXA microscope. An electron beam is focused on a luminescent film. A specimen is put on the luminescent film directly. The inset in Fig. 1(a) shows magnified image of the luminescent film and the specimen. Nanometric light source is excited in the luminescent film by the focused electron beam. The nanometric light source illuminates the specimen, and the scattered or transmitted radiation is detected with a photomultiplier tube (PMT). The light source is scanned by scanning of the focused electron beam in order to construct on image. Figure 1(b) shows a luminescence image of the cells acquired with the EXA microscope, and Fig. 1(c) shows a phase contrast microscope image. Cells were observed in culture solution without any treatments, such as fixation and drying. The shape of each cell was clearly recognized and some bright spots were observed in cells. We believe that the bright spots indicated with arrows were auto-fluorescence of intracellular granules and light- grey regions were auto-fluorescence of cell membranes. It is clearly demonstrated that the EXA microscope is useful tool for observation of living biological cells in physiological conditions.jmicro;63/suppl_1/i16/DFU090F1F1DFU090F1Fig. 1.(a) Optical setup of EXA microscpe, and observation results of of living MARCO-expressing CHO cells with (b) EXA microscope and (c) phase contrast microscope. We proposed the EXA microscope as a technique with high spatial resolution beyond the diffraction limit of light. A spatial resolution greater than 100 nm was achieved for the EXA microscope and the dynamic behavior of moving nanoparticles in water was observed by time lapse imaging. We also demonstrated luminescence image of living cells in culture solution without any treatments.
    Microscopy (Oxford, England). 11/2014; 63 Suppl 1:i16.
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    ABSTRACT: We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam. Electron beam excitation enables detailed observations on the nanometer scale. Real-time live-cell observation is also possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation. In this study, we demonstrated observation of cellular components by autofluorescence excited with a focused electron beam and performed dynamic observations of intracellular granules. Since autofluorescence is associated with endogenous substances in cells, this microscope can also be used to investigate the intrinsic properties of organelles.
    Biomedical Optics Express 02/2014; 5(2):378-86. · 3.18 Impact Factor
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    ABSTRACT: Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.
    ChemPhysChem 01/2014; · 3.35 Impact Factor
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    ABSTRACT: We present an Electron-beam-eXcitation-Assisted (EXA) optical microscope with a nanometric illumination light source consisting of red cathode luminescence (CL) lights emitted by a Y2O3:Eu3+ phosphor thin film excited by a high-energy focused electron beams. Phosphor films a few hundred nanometers thick were fabricated on 50-nm Si3N4 membranes using electron beam evaporation. The film preparation conditions for brighter CL emissions were examined in terms of the post-annealing temperatures and film thickness. We succeeded in spatially resolving gold nanoparticles with average diameter of 100 nm. The observations proved that the microscope has a spatial resolution higher than the diffraction limits.
    Optical Materials Express 01/2014; 4(1). · 2.92 Impact Factor
  • JSAP-OSA Joint Symposia; 09/2013
  • JSAP-OSA Joint Symposia; 09/2013
  • W. Inami, Y. Nawa, Y. Kawata
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    ABSTRACT: We present superresolving optical imaging system and demonstrate the observation of biological specimens without any stained process. The electron-beam excited assisted (EXA) optical microscope has a few tens nanometer spatial resolution laterally and is possible to observe dynamic behaviors of specimens in various surroundings such as air or liquids. In the EXA-microscope, a nano-light source in a few nanometers size is excited by focused electron beam in an emission layer. An electron beam can be focused to a spot size as small as 1 nanometer in diameter. The EXA-microscope enables to observe optical constants such as absorption, refractive index, polarization properties, and its dynamic behaviors in nanometer scale. We also have developed a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. In the microscope, fluorescent materials are directly excited with a focused electron beam. In this paper we present the evaluation result of resolution and observation results of living HeLa cells with high resolution. We have successfully observed fine structures of the cells without any stain process. This is first demonstration of observation of intercellular granules in HeLa cells.
    QiR (Quality in Research), 2013 International Conference on; 01/2013
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    ABSTRACT: We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.
    Optics Express 02/2012; 20(5):5629-35. · 3.55 Impact Factor
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    ABSTRACT: We propose a new type of scanning optical microscope which has a few tens nanometer spatial resolution laterally and is possible to observe dynamic behaviors of a specimen in various surroundings.
    Novel Techniques in Microscopy; 04/2011