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. A Nernstian-like pH response with a pH sensitivity of 53.83 mV/pH and linearity of 96.15% was obtained. The spatial resolution of the imaging system was evaluated by applying a photoresist to the sensing surface of the ion-sensor substrate. A spatial resolution of 216 nm was obtained. We achieved a substantially higher spatial resolution than that reported in the LAPS systems.

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. Our technique can observe both high-resolution surface images and the diffraction-limited three-dimensional structure by scanning a near-field light source on a single plane.

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. We found that nucleic acid and mitochondria in these label-free MC3T3-E1 cells quite strongly emit the autofluorescence as a result of DUV-SPR. Yeast cells are also deposited on the aluminum thin film. Tryptophan and mitochondrial nicotinamide adenine dinucleotide (NADH) in the yeast cells are subsequently excited, and their autofluorescence is spectrally analyzed in the UV region. On the basis of these results, we conclude that DUV-SPR constitutes a promising technique for the acquisition of highly sensitive autofluorescence images of various organelles in the cells.

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.

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. The resultant spectra enabled the dispersion relationship of Al-SPR in the FUV and DUV regions to be obtained. In the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on the Al film, the angle and wavelength of the SPR became larger and longer, respectively, compared to those in air. These shifts correspond well with the results of simulations performed using Fresnel equations.

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. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of adult newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo.

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.

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. 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.

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. We covalently coupled carboxyl groups on silicon nitride membranes, and measured the contact angles of water droplets on the surfaces to evaluate the hydrophilicity. We cultured HeLa cells on the coated membranes and evaluated stretch of the cell. Cell migration and confluence were observed on the coated silicon nitride films. We also demonstrated preliminary observation result with direct electron beam excitation-assisted optical microscope.

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.

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. However, it is also attributed to an increase in coupling efficiency with penetration depth and also to an increase in light extraction efficiency by grading the refractive indices at the heterostructure.

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.

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster.

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.

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. We have demonstrated controlled beam positioning capability to selectively irradiate 500 nm size structure with a point electron beam. We determined beam irradiation parameters that did not cause irreversible plasma membrane perforation after beam exposure and the irradiation caused intracellular Ca2+ elevation in an irradiated neuronal cell. Since the neuronal cell express fine subcellular structures such as neurites, we tried to position a beam on the structure and observed a Ca2+ wave originated from the intended point, which showed that our system had enough selectivity to target a subcellular structure. Point electron beam exposure is expected to be employed for various cellular stimulation protocols, and this enables the investigation of the biological functions of subcellular domains.

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. She had been treated for dermatomyositis, diabetes mellitus, and right parotid tumor for 3 years. Visual acuity was 0.1 OD and counting fingers OS. Slit-lamp examination showed grade 4 (Emery-Little classification) nuclear cataract in both eyes and keratoprecipitates and tan vitreous opacity in the left eye. Fundoscopy details were unclear except for a vaguely observable optic nerve head due to yellow-brown vitreous opacity, which we judged as an old vitreous hemorrhage. Phacovitrectomy was performed and almost total retinal detachment was found, except for a part of the superior periphery. Since no retinal break was found and a wide range of thin membrane-like tissue was found on the surface of the retina, the surgeon suspected primary IOL and performed unplanned biopsy. The peripheral vitreous was collected as a sample, and then the subretinal fluid was collected through an intentional break to prevent mixing with other fluids. The subretinal strand was gently removed and collected. Cytology showed class III, the IL10/IL6 ratio was low, and AIGHR was positive. Postoperatively, fundus autofluorescence showed no abnormality, no leakage was observed on fluorescein and indocyanine green angiography, and the location of typical infiltration lesions under the retina was unclear. There were no positive findings on systemic examinations and a diagnosis of primary IOL was made. The main symptoms of this case were vitreous opacity and exudative retinal detachment, and AIGHR using subretinal fluid was useful for diagnosis.

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. Furthermore, the gas in the bubble can be estimated from the CL emission spectrum. We have measured characteristics such as bubble size and particle number density. Also, the CL spectra has shown that the ultrafine bubbles contained nitrogen.

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. Fundus examination and optical coherence tomography showed SRD and RS in connection with the optic disc. The best-corrected logarithm of the minimum angle of resolution (logMAR) VA was 0.7. INTERVENTIONS: The patient underwent JPL treatment combined with pars plana vitrectomy. During surgery, posterior vitreous detachment and tamponade were created with sulfur hexafluoride. OUTCOMES: After surgery, SRD (and subsequently RS) gradually reduced and had completely disappeared at 31 months. VA gradually improved and was 0.0 (logMAR) at 28 months. The analysis of the mean macular thickness of the central 3-mm diameter showed that the macula thickness recovered to 300 µm at 17 months postoperatively. Retinal sensitivity began to improve at 24 months postoperatively and had increased at 48 months postoperatively. LESSONS: In conclusion, vitrectomy with JPL treatment for ODP-M had a favorable anatomical outcome as well as a long-term functional outcome. These findings provide useful information for clinicians who are planning a therapeutic strategy, including the choice of surgical procedure for ODP-M.

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.