Wataru Inami

Electron beam excitation assisted optical microscope with ultra-high resolution

We propose electron beam excitation assisted optical microscope, 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.

Fluorescence enhancement with deep-ultraviolet surface plasmon excitation

We report the experimental demonstration of fluorescence enhancement in fluorescent thin film using surface plasmon excitation in deep-ultraviolet (deep-UV) region. Surface plasmon resonance in deep-UV is excited on aluminum thin film in the Kretschmann-Raether geometry. Considering the oxidation thickness of aluminum, the experimentally measured incident angle dependence of reflectance show good agreement with Fresnel theory. Surface plasmon resonance was excited at the incident angle of 49 degrees for 266 nm p-polarized excitation light on the film of 18 nm-thick aluminum with 6.5 nm-thick alumina. Fluorescence of CdS quantum dots coated on this aluminum film was enhanced to 18-fold in intensity by the surface plasmon excitation.

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.

Deep-ultraviolet light excites surface plasmon for the enhancement of photoelectron emission

We show deep ultraviolet (DUV) light excitation of surface plasmon resonance (SPR) (DUV-SPR) with aluminum (Al) film. DUV-SPR has higher energy than that of visible light opens many applications, such as enhancement of photoelectron emission from metal surface, autofluorescence imaging of biological specimens, and laser ablation with high energy photons. We demonstrated the enhancement of photoelectron emission by DUV-SPR and analyzed protection layer to avoid oxidation of aluminum surface with enhancement of electric field kept as much as possible. The photoelectron emission from the aluminum surface was enhanced nine times with the excitation of SPR.

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.

The newt reprograms mature RPE cells into a unique multipotent state for retinal regeneration.

The reprogramming of retinal pigment epithelium (RPE) cells in the adult newt immediately after retinal injury is an area of active research for the study of retinal disorders and regeneration. We demonstrate here that unlike embryonic/larval retinal regeneration, adult newt RPE cells are not directly reprogrammed into retinal stem/progenitor cells; instead, they are programmed into a unique state of multipotency that is similar to the early optic vesicle (embryo) but preserves certain adult characteristics. These cells then differentiate into two populations from which the prospective-neural retina and -RPE layers are formed with the correct polarity. Furthermore, our findings provide insight into the similarity between these unique multipotent cells in newts and those implicated in retinal disorders, such as proliferative vitreoretinopathy, in humans. These findings provide a foundation for biomedical approaches that aim to induce retinal self-regeneration for the treatment of RPE-mediated retinal disorders.

Enhanced multicolor fluorescence in bioimaging using deep-ultraviolet surface plasmon resonance

Enhanced multicolor fluorescence has been achieved using deep-ultraviolet surface plasmon resonance (DUV-SPR) on an aluminum thin film using the Kretschmann configuration. The film thickness and the incident angle of the light were optimized by calculations using the Fresnel equations. The presence of a surface oxide layer was also considered in the calculations. Experimental measurements showed that DUV-SPR led to a strong enhancement of the fluorescence intensity from both quantum dots and dye-labeled cells.

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.

Analysis of electron and light scattering in a fluorescent thin film by combination of Monte Carlo simulation and finite-difference time-domain method

We analyzed light intensity distributions in a subwavelength fluorescent film, which was excited by a focused electron beam. We have developed an analyzing method using Monte Carlo simulation and the finite-difference time-domain (FDTD) method. Electron scattering and trajectories were calculated by Monte Carlo simulation. Propagation and scattering of light excited with the electrons was calculated by FDTD method. A nanometric light spot was formed on the fluorescent film surface and its light intensity and its full width at half maximum (FWHM) were evaluated. We discuss the intensity and the FWHM dependence on the thickness of the fluorescent thin film and the acceleration voltage of an incident electron beam.

Plasmonic nanofocusing using a metal-coated axicon prism

We propose an excitation method for the localization of photons at the apex of a metal coated axicon prism. The cone angle of the prism and the metallic film thickness are designed to match the excitation conditions for surface plasmons. The plasmons propagate along the sides of the prism and converge at its apex. The resulting nanofocusing was investigated by simulating the intensity distributions around the apex of the prism using a finite-difference time-domain algorithm. For incident radial polarization, a localized and field enhanced spot is generated by the constructive interference of surface plasmons.