Ovariectomy Increases Circulating Retinol-Binding Protein Concentrations Independently of Sex-Dependent Differences in Retinol Concentrations in Rats.

BACKGROUND: There is a sex-dependent difference in blood retinol and RBP concentrations, and plasma RBP is associated with insulin resistance. OBJECTIVES: We aimed to clarify sex-dependent variations in body concentrations of retinol and RBPs and their association with sex hormones in rats. METHODS: Plasma and liver retinol concentrations and hepatic mRNA and plasma concentrations of RBP4 were analyzed in 3- and 8-wk-old male and female Wistar rats before and after sexual maturity (experiment 1) and in orchiectomized male Wistar rats (experiment 2) and ovariectomized female Wistar rats (experiment 3). Furthermore, the mRNA and protein concentrations of RBP4 in adipose tissue were measured in ovariectomized female rats (experiment 3). RESULTS: There were no sex-dependent differences in liver retinyl palmitate and retinol concentrations; however, the plasma retinol concentration was significantly higher in male rats than that in female rats after sexual maturity. Furthermore, the plasma retinol concentrations did not differ between the ovariectomized or orchiectomized rats and the control rats. Plasma Rbp4 mRNA concentrations were higher in male rats than those in female rats but not in castrated and control rats, a change consistent with plasma retinol concentration. Plasma RBP4 concentrations were also higher in male rats than those in female rats; however, unlike liver Rbp4 gene expression, plasma RBP4 concentrations were 7-fold higher in the ovariectomized rats than those in the control rats. Moreover, the Rbp4 mRNA concentrations in inguinal white adipose tissue was significantly higher in the ovariectomized rats than those in the control rats and correlated with plasma RBP4 concentrations. CONCLUSIONS: Hepatic Rbp4 mRNA is higher in male rats through a sex hormone-independent mechanism, which may contribute to sex differences in blood retinol concentrations. Furthermore, ovariectomy leads to an increase in adipose tissue Rbp4 mRNA and blood RBP4 concentrations, which may contribute to insulin resistance in ovariectomized rats and postmenopausal women.

Simple autofocusing method by image processing using transmission images for large-scale two-photon lithography.

We propose a simple autofocusing technique that can be introduced into conventional two-photon lithography systems without additional devices. Autofocusing is achieved by image processing using transmission images of photopolymerized voxels. The signal-to-noise ratio of transmission images was improved by optimal low-pass filtering to detect voxels in them. The focal point was detected with an accuracy of about 250 nm from the difference images. Further, we demonstrated mass-fabrication of a 5 × 5 spiral square array with an area of 900 × 900 µm2 using this method. The method has potential application in constructing low-cost, compact and versatile two-photon lithography apparatus.

Multi-scale laser direct writing of conductive metal microstructures using a 405-nm blue laser.

A multi-scale direct writing method for metal microstructures is proposed and demonstrated. In this study, metal structures were created in a gelatin matrix containing silver nitrate by photoreduction using a 405-nm blue laser. The influence of concentrations of materials in the sample solution was evaluated by measuring the conductivity of the fabricated microstructures. The fabrication line width could be controlled by changing the laser scanning speed. A network structure was also observed, which possibly helps in increasing the microstructure's conductivity. Finally, we demonstrated multi-scale drawing by using objective lenses with different numerical apertures. Our method can result in new possibilities for conductive metal direct writing.

Cellular force assay detects altered contractility caused by a nephritis-associated mutation in nonmuscle myosin IIA.

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II (NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney (HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease.

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.

Fatty acid amides present in Camembert cheese improved cognitive decline after oral administration in mice.

Herein, we investigated the effects of Camembert cheese (CC) and its fatty acid contents on cognitive function in mice by employing the object recognition test to evaluate hippocampus-dependent memory. Orally administered CC improved the cognitive decline induced by a high-fat diet. Next, we focused on myristamide (MA), oleamide, and stearamide, which are fatty acid amides produced during the fermentation process of CC. We found that oral administration of MA improved cognitive decline. Notably, an improvement was not observed using myristic acid, a free fatty acid that is not amidated. Thus, fatty acid amidation may contribute to the physiological activity. Moreover, we investigated changes in gene expression related to neurogenesis in the hippocampus. After MA administration, mRNA expression analysis indicated that MA increased hippocampal brain-derived neurotrophic factor expression.

Bubble Printing of Liquid Metal Colloidal Particles for Conductive Patterns.

Bubble printing is a patterning method in which particles are accumulated by the convection of bubbles generated by laser focusing. It is attracting attention as a method that enables the high-speed, high-precision patterning of various micro/nanoparticles. Although the bubble printing method is used for metallic particles and organic particles, most reports have focused on the patterning of solid particles and not on the patterning of liquid particles. In this study, liquid metal wiring patterns were fabricated using a bubble printing method in which eutectic gallium‒indium alloy (EGaIn) colloidal particles (≈diameter 0.7 µm) were fixed on a glass substrate by generating microbubbles through heat generation by focusing a femtosecond laser beam on the EGaIn colloidal particles. The wiring was then made conductive by replacing gallium oxide, which served as a resistance layer on the surface of the EGaIn colloidal particles, with silver via galvanic replacement. Fine continuous lines of liquid metal colloids with a line width of 3.4 µm were drawn by reducing the laser power. Liquid metal wiring with a conductivity of ≈1.5 × 105 S/m was formed on a glass substrate. It was confirmed that the conductivity remained consistent even when the glass substrate was bent to a curvature of 0.02 m-1.

High-resolution microscopy for biological specimens via cathodoluminescence of Eu- and Zn-doped Y2O3 nanophosphors.

High-resolution microscopy for biological specimens was performed using cathodoluminescence (CL) of Y(2)O(3):Eu, Zn nanophosphors, which have high CL intensity due to the incorporation of Zn. The intensity of Y(2)O(3):Eu nanophosphors at low acceleration voltage (3 kV) was increased by adding Zn. The CL intensity was high enough for imaging even with a phosphor size as small as about 30 nm. The results show the possibility of using CL microscopy for biological specimens at single-protein-scale resolution. CL imaging of HeLa cells containing laser-ablated Y(2)O(3):Eu, Zn nanophosphors achieved a spatial resolution of a few tens of nanometers. Y(2)O(3):Eu, Zn nanophosphors in HeLa cells were also imaged with 254 nm ultraviolet light excitation. The results suggest that correlative microscopy using CL, secondary electrons and fluorescence imaging could enable multi-scale investigation of molecular localization from the nanoscale to the microscale.

Fabrication of Flexible Wiring with Intrinsically Conducting Polymers Using Blue-Laser Microstereolithography.

Recently, flexible devices using intrinsically conductive polymers, particularly poly(3,4-ethylenedioxythiophene) (PEDOT), have been extensively investigated. However, most flexible wiring fabrication methods using PEDOT are limited to two-dimensional (2D) fabrication. In this study, we fabricated three-dimensional (3D) wiring using the highly precise 3D printing method of stereolithography. Although several PEDOT fabrication methods using 3D printing systems have been studied, few have simultaneously achieved both high conductivity and precise accuracy. In this study, we review the post-fabrication process, particularly the doping agent. Consequently, we successfully fabricated wiring with a conductivity of 16 S cm-1. Furthermore, flexible wiring was demonstrated by modeling the fabricated wiring on a polyimide film with surface treatment and creating a three-dimensional fabrication object.

3D-Printed Micro-Tweezers with a Compliant Mechanism Designed Using Topology Optimization.

The development of handling technology for microscopic biological samples such as cells and spheroids has been required for the advancement of regenerative medicine and tissue engineering. In this study, we developed micro-tweezers with a compliant mechanism to manipulate organoids. The proposed method combines high-resolution microstereolithography that uses a blue laser and topology optimization for shape optimization of micro-tweezers. An actuation system was constructed using a linear motor stage with a force control system to operate the micro-tweezers. The deformation of the topology-optimized micro-tweezers was examined analytically and experimentally. The results verified that the displacement of the tweezer tip was proportional to the applied load; furthermore, the displacement was sufficient to grasp biological samples with an approximate diameter of several hundred micrometers. We experimentally demonstrated the manipulation of an organoid with a diameter of approximately 360 µm using the proposed micro-tweezers. Thus, combining microstereolithography and topology optimization to fabricate micro-tweezers can be potentially used in modifying tools capable of handling various biological samples.

Additive Manufacturing of Micromanipulator Mounted on a Glass Capillary for Biological Applications.

In this study, a three-dimensional (3D) micromanipulator mounted on a glass capillary is developed for handling biological samples, such as multicellular spheroids and embryos. To fabricate the micromanipulator, we developed an additive manufacturing system based on high-resolution microstereolithography using a 405-nm blue laser. The fabrication system makes it possible to fabricate 3D microstructures on a glass capillary with 2.5 µm lateral resolution and 25 µm layer thickness. We also demonstrated the capture and release of a spheroid with the micromanipulator fabricated using our additive manufacturing system. We showed that spheroids can be easily handled by a simple operation with minimal damage using a cage-like multiple finger structure. Additive manufacturing of tailor-made micromanipulators mounted on a glass capillary will be useful in biological and tissue engineering research.

Excitation of erbium-doped nanoparticles in 1550-nm wavelength region for deep tissue imaging with reduced degradation of spatial resolution.

Rare-earth-doped nanoparticles are one of the emerging probes for bioimaging due to their visible-to-near-infrared (NIR) upconversion emission via sequential single-photon absorption at NIR wavelengths. The NIR-excited upconversion property and high photostability make this probe appealing for deep tissue imaging. So far, upconversion nanoparticles include ytterbium ions (Yb3 + ) codoped with other rare earth ions, such as erbium (Er3 + ) and thulium (Tm3 + ). In these types of upconversion nanoparticles, through energy transfer from Yb3 + excited with continuous wave light at a wavelength of 980 nm, upconversion emission of the other rare earth dopants is induced. We have found that the use of the excitation of Er3 + in the 1550-nm wavelength region allows us to perform deep tissue imaging with reduced degradation of spatial resolution. In this excitation­emission process, three and four photons of 1550-nm light are sequentially absorbed, and Er3 + emits photons in the 550- and 660-nm wavelength regions. We demonstrate that, compared with the case using 980-nm wavelength excitation, the use of 1550-nm light enables us to moderate degradation of spatial resolution in deep tissue imaging due to the lower light scattering coefficient compared with 980-nm light. We also demonstrate that live cell imaging is feasible with this 1550 nm excitation.

3D Shape Reconstruction of 3D Printed Transparent Microscopic Objects from Multiple Photographic Images Using Ultraviolet Illumination.

We propose and demonstrate a simple, low-cost, three-dimensional (3D) shape acquisition method for transparent 3D printed microscopic objects. Our method uses ultraviolet (UV) illumination to obtain high-contrast silhouette images of transparent 3D printed polymer objects. Multiple silhouette images taken from different viewpoints make it possible to reconstruct the 3D shape of this transparent object. A 3D shape acquisition system consisting of a UV light-emitting diode, charge-coupled device camera and a rotation stage was constructed and used to successfully reconstruct the 3D shape of a transparent bunny model produced using micro-stereolithography. In addition, 3D printed pillar array models, with different diameters on the order of several hundred micrometers, were reconstructed. This method will be a promising tool for the 3D shape reconstruction of transparent 3D objects on both the micro- and macro-scale by changing the imaging lens.

Multispectral Emissions of Lanthanide-Doped Gadolinium Oxide Nanophosphors for Cathodoluminescence and Near-Infrared Upconversion/Downconversion Imaging.

Comprehensive imaging of a biological individual can be achieved by utilizing the variation in spatial resolution, the scale of cathodoluminescence (CL), and near-infrared (NIR), as favored by imaging probe Gd2O3 co-doped lanthanide nanophosphors (NPPs). A series of Gd2O3:Ln3+/Yb3+ (Ln3+: Tm3+, Ho3+, Er3+) NPPs with multispectral emission are prepared by the sol-gel method. The NPPs show a wide range of emissions spanning from the visible to the NIR region under 980 nm excitation. The dependence of the upconverting (UC)/downconverting (DC) emission intensity on the dopant ratio is investigated. The optimum ratios of dopants obtained for emissions in the NIR regions at 810 nm, 1200 nm, and 1530 nm are applied to produce nanoparticles by the homogeneous precipitation (HP) method. The nanoparticles produced from the HP method are used to investigate the dual NIR and CL imaging modalities. The results indicate the possibility of using Gd2O3 co-doped Ln3+/Yb3+ (Ln3+: Tm3+, Ho3+, Er3+) in correlation with NIR and CL imaging. The use of Gd2O3 promises an extension of the object dimension to the whole-body level by employing magnetic resonance imaging (MRI).

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.

Rare-earth-doped nanophosphors for multicolor cathodoluminescence nanobioimaging using scanning transmission electron microscopy.

We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence(CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3∶Eu, Y2O3∶Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light.Y2O3∶Tb and Y2O3∶Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared,and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since theRE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.

Y2O3:Tm,Yb nanophosphors for correlative upconversion luminescence and cathodoluminescence imaging.

We present a phosphor nanoparticle that shows both upconversion luminescence (UCL) and cathodoluminescence (CL). With this particle, low-autofluorescence, deep-tissue and wide-field fluorescence imaging can be achieved with nanometer-order high-spatial-resolution imaging. We synthesized Y2O3:Tm,Yb nanophosphors that emit visible and near-infrared UCL under 980 nm irradiation and blue CL via electron beam excitation. The phosphors were applied to fluorescent imaging of HeLa cells. The photostability of the phosphors was superior to that of a conventional organic dye. We show that after uptake by HeLa cells, the particles can be imaged with SEM and CL contrast in a cellular section. This indicates that correlative UCL and CL imaging of biological samples could be realized.