Non-invasive nanoscale imaging of protein micro- and nanocrystals for screening crystallization conditions.

Crystallography has been the routine technique for studying high-resolution structures of proteins for over five decades. A major bottleneck in structure determination of macromolecules is obtaining crystals of a size and quality suitable for single-crystal X-ray crystallography experiments. Many challenging proteins either fail to grow into crystals or fail to grow into crystals of a size suitable for obtaining high-resolution structures using conventional X-ray crystallography.

Development of a localized surface plasmon-enhanced electron beam-pumped nanoscale light source for electron beam excitation-assisted optical microscopy.

We have demonstrated localized surface plasmon (LSP)-enhanced cathodoluminescence (CL) from an atomic layer deposition (ALD)-grown Al2O3/ZnO/Al2O3 heterostructure to develop a bright nanometer-scale light source for an electron beam excitation-assisted (EXA) optical microscope. Three types of metals, Ag, Al, and Au, were compared, and an 181-fold enhancement of CL emission was achieved with Ag nanoparticles (NPs), with the plasmon resonance wavelength close to the emission wavelength energy of ZnO. The enhanced emission is plausibly attributed to LSP/exciton coupling.

High resolution imaging of ultrafine bubbles in water by Atmospheric SEM-CL.

Various analytical methods such as high-resolution observation of ultrafine bubbles in water are required to clarify the mechanisms and interrelationships of various effects brought about by ultrafine bubbles. In this study, we used atmospheric scanning electron microscopy-cathodoluminescence (ASEM-CL) method for observing ultrafine bubbles in water. ASEM can observe samples in water, and the fine electron beam provides high spatial resolution.

Development of a direct point electron beam exposure system to investigate the biological functions of subcellular domains in a living biological cell.

Electron microscopy studies have demonstrated that the diameter of a focused electron beam is small enough to probe or manipulate subcellular domains of a single biological cell. Here, we report the development of a direct point electron beam irradiation system to investigate the biological functions of subcellular domains in a living cell. Subcellular structures of a single living cell cultured on a thin film can be selectively irradiated by the point electron beam generated by our system.

High Spatial Resolution Ion Imaging by Focused Electron-Beam Excitation with Nanometric Thin Sensor Substrate.

We developed a high spatially-resolved ion-imaging system using focused electron beam excitation. In this system, we designed a nanometric thin sensor substrate to improve spatial resolution. The principle of pH measurement is similar to that of a light-addressable potentiometric sensor (LAPS), however, here the focused electron beam is used as an excitation carrier instead of light.

Depth structure analysis by surface scanning in near-field microscopes.

High-resolution imaging of the surfaces of samples can be performed using near-field optical microscopes by scanning a small light spot; however, structures located deep beneath cannot be observed because the light spot spreads in three directions. In this study, we propose an observation technique for near-field optical microscopes that can obtain depth information within the resolution of the diffraction limit of light by analyzing interference patterns formed with divergent incident light and scattered light from a sample. We analyze depth structures by evaluating correlation coefficients between observed interference patterns and calculated reference patterns.

Enhanced Surface Plasmon Resonance Wavelength Shifts by Molecular Electronic Absorption in Far- and Deep-Ultraviolet Regions.

In this study, surface plasmon resonance (SPR) wavelength shifts due to molecular electronic absorptions in the far-ultraviolet (FUV, < 200 nm) and deep-ultraviolet (DUV, < 300 nm) regions were investigated by attenuated total reflectance (ATR) spectroscopy. Due to the strong absorption in the DUV region, N,N-dimethylformamide (DMF) significantly increased the SPR wavelength shift of Al film. On the other hand, no such shift enhancement was observed in the visible region for Au film because DMF does not have absorbance compared to non-absorbing materials such as water and alcohols.

Ultrashort laser based two-photon phase-resolved fluorescence lifetime measurement method.

This paper presents a two-photon phase-resolved fluorescence-lifetime measurement method based on the use of an ultrashort pulse laser. The proposed method also involves the use of a lock-in amplifier to control the phase difference between the reference and fluorescence signals, thereby facilitating the use of an alternative method for determining fluorescence lifetimes. Verification of the fluorescence lifetimes as measured in this study was performed using rhodamine B and a cellular thermoprobe as samples.

Cell stimulation by focused electron beam of atmospheric SEM.

Cell stimulation has been performed with a focused electron beam. To protect the live cells from the vacuum environment of the electron beam, the beam irradiated the ambient cells via a thin film. In this way, the cells were electrically stimulated with nanometre resolution in a non-contact process.

Numerical simulations of partially coherent illumination for multi-scattering phase objects via a beam propagation method.

Many studies have performed imaging under partially coherent illumination. However, to the best of our knowledge, there are no imaging methods for complicated objects such as cell colonies, which are large and diffract light multiple times. In this paper, we propose an image calculation method for the partially coherent illumination of a large-scale three-dimensional multi-diffractive object.

Quantitative phase imaging by optimized asymmetric illumination.

We have presented a simple approach for quantitative phase imaging by optimizing asymmetric illumination of a conventional microscope. With this illumination, the light intensity modulation accompanying refraction at the surface profile of phase objects occurs, and "phase-gradient information" can be derived by detecting it. Two images with phase-gradient information on different axes are converted into the two-dimensional phase distribution of the specimen by introducing the phase-gradient transfer function, which is the intensity change due to refraction by the phase-gradient of a specimen.

Far- and deep-ultraviolet surface plasmon resonance sensors working in aqueous solutions using aluminum thin films.

Surface plasmon resonance (SPR) sensors detect refractive index changes on metal thin films and are frequently used in aqueous solutions as bio- and chemical-sensors. Recently, we proposed new SPR sensors using aluminum (Al) thin films that work in the far- and deep-ultraviolet (FUV-DUV, 120-300 nm) regions and investigated SPR properties by an attenuated total reflectance (ATR) based spectrometer. The FUV-DUV-SPR sensors are expected to have three advantages compared to visible-SPR sensors: higher sensitivity, material selectivity, and surface specificity.

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.

Cell structure imaging with bright and homogeneous nanometric light source.

Label-free optical nano-imaging of dendritic structures and intracellular granules in biological cells is demonstrated using a bright and homogeneous nanometric light source. The optical nanometric light source is excited using a focused electron beam. A zinc oxide (ZnO) luminescent thin film was fabricated by atomic layer deposition (ALD) to produce the nanoscale light source.

Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label-Free Cells by Deep-Ultraviolet Excitation.

We demonstrate the observation of organelles in label-free cells on an aluminum thin film using deep-ultraviolet surface plasmon resonance (DUV-SPR). In particular, the Kretschmann configuration is used for the excitation of DUV-SPR. MC3T3-E1 cells are directly cultured on the aluminum thin film, and DUV-SPR leads to autofluorescence of in the label-free MC3T3-E1.

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.

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.

Cell culture on hydrophilicity-controlled silicon nitride surfaces.

Cell culture on silicon nitride membranes is required for atmospheric scanning electron microscopy, electron beam excitation assisted optical microscopy, and various biological sensors. Cell adhesion to silicon nitride membranes is typically weak, and cell proliferation is limited. We increased the adhesion force and proliferation of cultured HeLa cells by controlling the surface hydrophilicity of silicon nitride membranes.

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.

Fabrication of bright and thin Zn₂SiO₄ 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.

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.

High resolution fluorescent bio-imaging with electron beam excitation.

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.

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.