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.

A Case of Intraocular Lymphoma Diagnosed by Subretinal Fluid Biopsy.

Although intraocular lymphoma (IOL) mainly has have vitreous opacity and subretinal infiltration, its clinical symptoms are diverse. We report a case of IOL that mainly showed exudative retinal detachment in which analysis of IgH gene rearrangement (AIGHR) of the collected subretinal fluid sample was useful for diagnosis. A 77-year-old woman developed decreased left visual acuity for 1 month.

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.

Quantitative assessment of macular function after surgery for optic disc pit maculopathy: A case report.

Rationale: We describe a case of optic disc pit maculopathy (ODP-M) in which vitrectomy with juxtapapillary laser (JPL) treatment led to the reattachment of retinoschisis (RS) as well as serous retinal detachment (SRD).

Patient Concerns: An 80-year-old man complained of distorted vision and decreased visual acuity (VA) in his left eye for 12 months.

Diagnosis: We conducted quantitative functional evaluation on the area of RS and SRD using the Humphrey visual field analyzer.

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.

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.

Direct optical measurements of far- and deep-ultraviolet surface plasmon resonance with different refractive indices.

The surface plasmon resonance (SPR) of Al thin films was investigated by varying the refractive index of the environment near the films in the far-ultraviolet (FUV, 120-200 nm) and deep-ultraviolet (DUV, 200-300 nm) regions. An original FUV-DUV spectrometer that adopts an attenuated total reflectance (ATR) system was used. The measurable wavelength range was down to the 180 nm, and the environment near the Al surface could be controlled.

Turning the fate of reprogramming cells from retinal disorder to regeneration by Pax6 in newts.

The newt, a urodele amphibian, has an outstanding ability- even as an adult -to regenerate a functional retina through reprogramming and proliferation of the retinal pigment epithelium (RPE) cells, even though the neural retina is completely removed from the eye by surgery. It remains unknown how the newt invented such a superior mechanism. Here we show that disability of RPE cells to regenerate the retina brings about a symptom of proliferative vitreoretinopathy (PVR), even in the newt.

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.

Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt.

The adult newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown.

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.