Masahiro Fukuta

Fabrication of bright and thin Zn 2 SiO 4 luminescent film for electron beam excitation-assisted optical microscope

We fabricated a bright and thin Zn₂SiO₄ luminescent film to serve as a nanometric light source for high-spatial-resolution optical microscopy based on electron beam excitation. The Zn₂SiO₄ luminescent thin film was fabricated by annealing a ZnO film on a Si₃N₄ substrate at 1000 °C in N₂. 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 Zn₂SiO₄ luminescent thin film. This is the first report of the investigation and application of ZnO/Si₃N₄ annealed at a high temperature (1000 °C). The fabricated Zn₂SiO₄ film is expected to enable high-frame-rate dynamic observation with ultra-high resolution using our electron beam excitation-assisted optical microscopy.

Intensity distribution analysis of cathodoluminescence using the energy loss distribution of electrons

We present an intensity distribution analysis of cathodoluminescence (CL) excited with a focused electron beam in a luminescent thin film. The energy loss distribution is applied to the developed analysis method in order to determine the arrangement of the dipole locations along the path of the electron traveling in the film. Propagating light emitted from each dipole is analyzed with the finite-difference time-domain (FDTD) method. CL distribution near the film surface is evaluated as a nanometric light source. It is found that a light source with 30 nm widths is generated in the film by the focused electron beam. We also discuss the accuracy of the developed analysis method by comparison with experimental results. The analysis results are brought into good agreement with the experimental results by introducing the energy loss distribution.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

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.

Absorption contrast imaging beyond the diffraction limit with electron-beam excitation assisted optical microscope

We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 having absorption at UV wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film of an electron-beam excitation assisted (EXA) optical microscope. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal. The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA optical microscope is useful for analysis of growth of a crystal, bioimaging, and so on.

Label-free cellular structure imaging with 82 nm lateral resolution using an electron-beam excitation-assisted optical microscope.

We present label-free and high spatial-resolution imaging for specific cellular structures using an electron-beam excitation-assisted optical microscope (EXA microscope). Images of the actin filament and mitochondria of stained HeLa cells, obtained by fluorescence and EXA microscopy, were compared to identify cellular structures. Based on these results, we demonstrated the feasibility of identifying label-free cellular structures at a spatial resolution of 82 nm. Using numerical analysis, we calculated the imaging depth region and determined the spot size of a cathodoluminescent (CL) light source to be 83 nm at the membrane surface.

High spatial resolution absorption contrast imaging with electron-beam excitation assisted optical microscope

We present high spatial-resolution label-free imaging with an electron-beam excitation-assisted optical microscope (EXA microscope). The EXA microscope improves the spatial resolution down to 100 nm. To realize the high spatial resolution, a nanoscale optical spot is generated by irradiating a fluorescent thin film with a focused electron beam whose spot size is less than 10 nm. The size of the optical spot becomes smaller than the diffraction limited spot size and is reduced to about 100 nm, because the light emission is localized in nanometer-sized region. In this microscopy, it is not necessary to label a specimen for imaging beyond the diffraction limit of the light. The specimen stage is separated from the vacuum chamber of the scanning electron microscope by the fluorescent thin film and a specimen under atmospheric pressure can be imaged. We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 having absorption at UV wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film we deposited. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal measured with an atomic force microscope (AFM). The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA microscope is useful for analysis of growth of a crystal, bio-imaging, and so on.

Fabrication of ZnO luminescent films for nanometric light source of high-resolution optical microscope

We fabricated ZnO thin films for use as a light source of a high resolution optical microscope and characterized the properties of the films. The properties of the fabricated ZnO films were evaluated as the light source of the microscope. The cathodoluminescence intensity of the 100 nm thick ZnO film deposited on a Si3N4 film was enhanced significantly by high temperature annealing in N2. The microscope images of 200 nm diameter gold particles taken using as-deposited and annealed ZnO film indicated that annealed ZnO films can provide a higher signal-to-noise ratio and a higher frame rate than the as deposited ZnO film.

The calculation of intensity distribution of cathodoluminescence in fluorescent thin film

This study analyze the intensity distribution of CL generated in fluorescent thin film by combination of Monte Carlo method and finite difference time domain (FDTD) method. The Monte Carlo method analyzes the electron beam scattering in the fluorescent thin film and FDTD method analyzes the light propagation of CL. From analysis result, the spot size of CL at surface of 50 nm Y2O3: Eu3+ is 43 nm. The correlation coefficient between analysis result and experimental result is 0.997. Therefore, the analysis method combined Monte Carlo method and FDTD method has high accuracy.