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.

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.

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.

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.

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.

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.

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. The enhanced SPR wavelength shift, caused by the overlap between SPR and molecular resonance wavelengths in FUV-DUV region, is expected to result in high sensitivity for resonant materials.

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. In this study, we assume that the fluorescence decay was monoexponential in all cases. Rhodamine B was observed to exhibit an average fluorescence lifetime of 2.15 ns, whereas a temperature sensitivity of 1.39 ns C-1 over a temperature range of 33.79-37.2 °C was demonstrated for the cellular thermoprobe. These results validate the feasibility of the proposed method for accurate measurement of fluorescence lifetimes using a simple laser configuration.

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. The response of calcium ion concentration in a single HeLa cell after electron beam irradiation was examined. This technique has the potential to stimulate single ion channels, granules, and organelles.

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. However, in this study, it was revealed that the Al thin film on a quartz prism cannot be used as the FUV-DUV-SPR sensor in water solutions. This is because its SPR wavelength shifts to the visible region owing to the presence of water. On the other hand, the SPR wavelength of the Al thin film on the sapphire prism remained in the DUV region even in water. In addition, the SPR wavelength shifted to longer wavelengths with increasing refractive index on the Al thin film. These results mean that the Al thin film on the sapphire prism can be used as the FUV-DUV-SPR sensor in solutions, which may lead to the development of novel and sophisticated SPR sensors.

Spatial resolution and cathodoluminescence intensity dependence on acceleration voltage in electron beam excitation assisted optical microscopy using Y2O3:Eu3+ film.

This study presents relationship between acceleration voltage and spatial resolution of electron-beam assisted (EXA) optical microscope. The nanometric illumination light sources of the present EXA microscope was red-emitting cathodoluminescence (CL) in the Y2O3:Eu3+ thin film excited by focused electron beam. Our experimental results demonstrated that the spatial resolutions of the EXA microscope were higher as the acceleration voltage was higher. We managed to make images of the scattered gold particles with approximately 90 nm-resolutions at the voltages higher than 20 kV. The dependence of the spatial resolution on the acceleration voltage was explained by the distribution of simulated electron scattering trajectories in the luminescent thin film.

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. The ZnO film formed by ALD emitted the bright, homogeneous light, unlike that deposited by another method. The dendritic structures of label-free macrophage receptor with collagenous structure-expressing CHO cells were clearly visualized below the diffraction limit. The inner fiber structure was observed with 120 nm spatial resolution. Because the bright homogeneous emission from the ZnO film suppresses the background noise, the signal-to-noise ratio (SNR) for the imaging results was greater than 10. The ALD method helps achieve an electron beam excitation assisted microscope with high spatial resolution and high SNR.

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.

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. When Pax6, a transcription factor that is re-expressed in reprogramming RPE cells, is knocked down in transgenic juvenile newts, these cells proliferate but eventually give rise to cell aggregates that uniformly express alpha smooth muscle actin, Vimentin and N-cadherin, the markers of myofibroblasts which are a major component of the sub-/epi-retinal membranes in PVR. Our current study demonstrates that Pax6 is an essential factor that directs the fate of reprogramming RPE cells toward the retinal regeneration. The newt may have evolved the ability of retinal regeneration by modifying a mechanism that underlies the RPE-mediated retinal disorders.

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.

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.

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.

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. CL distribution near the film surface is evaluated as a nanometric light source. It is found that a light source with 30 nm widths is generated in the film by the focused electron beam. We also discuss the accuracy of the developed analysis method by comparison with experimental results. The analysis results are brought into good agreement with the experimental results by introducing the energy loss distribution.

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.