[Show abstract][Hide abstract] ABSTRACT: Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.
[Show abstract][Hide abstract] ABSTRACT: Intracellular structures of HeLa cells are observed using a direct electron beam excitation-assisted fluorescence (D-EXA) microscope. In this microscope, a silicon nitride membrane is used as a culture plate, which typically has a low biocompatibility between the sample and the silicon nitride surface to prevent the HeLa cells from adhering strongly to the surface. In this work, the surface of silicon nitride is modified to allow strong cell attachment, which enables high-resolution observation of intracellular structures and an increased signal-to-noise ratio. In addition, the penetration depth of the electron beam is evaluated using Monte Carlo simulations. We can conclude from the results of the observations and simulations that the surface modification technique is promising for the observation of intracellular structures using the D-EXA microscope.
No preview · Article · Aug 2015 · Biomedical Optics Express
[Show abstract][Hide abstract] ABSTRACT: We fabricated a bright and thin Zn<sub>2</sub>SiO<sub>4</sub> luminescent film to serve as a nanometric light source for high-spatial-resolution optical microscopy based on electron beam excitation. The Zn<sub>2</sub>SiO<sub>4</sub> luminescent thin film was fabricated by annealing a ZnO film on a Si<sub>3</sub>N<sub>4</sub> substrate at 1000 °C in N<sub>2</sub>. The annealed film emitted bright cathodoluminescence compared with the as-deposited film. The film is promising for nano-imaging with electron beam excitation-assisted optical microscopy. We evaluated the spatial resolution of a microscope developed using this Zn<sub>2</sub>SiO<sub>4</sub> luminescent thin film. This is the first report of the investigation and application of ZnO/Si<sub>3</sub>N<sub>4</sub> annealed at a high temperature (1000 °C). The fabricated Zn<sub>2</sub>SiO<sub>4</sub> film is expected to enable high-frame-rate dynamic observation with ultra-high resolution using our electron beam excitation-assisted optical microscopy.
[Show abstract][Hide abstract] ABSTRACT: High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.
[Show abstract][Hide abstract] ABSTRACT: A plastic scintillator film for use in an electron beam excitation-assisted (EXA) optical microscope is characterized. The thin film scatters an incident electron beam weakly and generates high intensity nanoscale luminescence excited by the beam spot. For
high spatial resolution and signal to noise, an EXA microscope requires a thin high-efficiency scintillator film. Homogeneous plastic scintillators with thicknesses ranging from 60 to 2800 nm were fabricated on silicon nitride via spin coating. The emission intensity was examined as a function of film thickness and the accelerating voltage of the incident electron beam. The emission wavelength can be tuned by
changing scintillator materials in the film matrix. To demonstrate a plastic scintillator film performance with an EXA microscope, time-lapse images of yeast cells were acquired.
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2014 · Microscopy (Oxford
[Show abstract][Hide abstract] ABSTRACT: We fabricated ZnO/SiN films for use as a light source of a high-resolution optical microscope and characterized the properties of the films, and demonstrated images obtained with the microscope using the fabricated ZnO/SiN films. A 100-nm-thick ZnO film deposited on a SiN film showed a much higher CL intensity than the SiN film, and it was enhanced by high-temperature annealing of the ZnO film. Electron beam excitation assisted optical microscope images of gold particles of 200nm diameter taken using the ZnO/SiN film and SiN indicated that the ZnO/SiN films can provide a higher signal-to-noise (S/N) ratio and a higher frame rate than the SiN film. It was shown that the dynamic observation of living cells becomes possible using the high-resolution optical microscope with a bright light source. Moreover, this fact promises that such optical microscope can contribute to progress in the medical and biological fields. (C) 2014 The Japan Society of Applied Physics
No preview · Article · Apr 2014 · Japanese Journal of Applied Physics
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Full-text · Article · Feb 2014 · Biomedical Optics Express
[Show abstract][Hide abstract] 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.
Preview · Article · Jan 2014 · Optical Materials Express
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.