Yasunori Nawa

Dynamic and high-resolution live cell imaging by direct electron beam excitation.

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.

Multi‐Color Imaging of Fluorescent Nanodiamonds in Living HeLa Cells Using Direct Electron‐Beam Excitation

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.

Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation

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.

Nanometric light spots of cathode luminescence in Y 2 O 3 :Eu 3+ phosphor thin films excited by focused electron beams as ultra-small illumination source for high-resolution optical microscope

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.

Formation of ZnO luminescent films on SiN films for light source of high-resolution optical microscope

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 200 nm 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.

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.

High-resolution, label-free imaging of living cells with direct electron-beam-excitation-assisted optical microscopy

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.

Carboxylic monolayer formation for observation of intracellular structures in HeLa cells with direct electron beam excitation-assisted fluorescence microscopy

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.

Evaluation of cell damage induced by electron beam

We evaluate the damage on living cells induced by electron beam irradiation. Cell viability is observed with a fluorescence microscope. Electron beam induced damage is evaluated by the comparison of fluorescence images obtained before and after the electron beam irradiation. After electron beam irradiation, a bleb related to cell injury was observed and cell viability signal was decreased on the irradiated cell.

Hela cell culturing on a hydrophilicity controlled silicon nitride surface

Cell culture on a silicon nitride membrane is required for atmospheric scanning electron microscopy, electron beam excitation-assisted optical microscopy, and various biological sensors. Cell adhesion to a silicon nitride membrane is weak, and the proliferation potential is also limited. Therefore, we considered that the adhesion force and proliferation potential of cultured cells might be increased by controlling the surface hy-drophilicity. In this study, we covalently coupled carboxyl groups on silicon nitride membranes to increase the hydrophilicity. We measured contact angles of water droplets on silicon nitride membrane surfaces to evaluate the hydrophilicity. Moreover, we successfully cultured HeLa cells on coated membranes. Cell migration and confluence were observed on the coated silicon nitride film.