Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English  

DOI： 10.1111/exd.14759

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, the knockdown of which significantly suppressed the upregulation of DPT, ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated fibroblasts were positive for senescence-associated β-galactosidase. Finally, transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

DOI： 10.1038/s42003-022-04408-3

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Layer V neurons in the primary motor cortex (M1) are important for motor skill learning. Since pretreatment of either CNQX or APV in rat M1 layer V impaired rotor rod learning, we analysed training-induced synaptic plasticity by whole-cell patch-clamp technique in acute brain slices. Rats trained for 1 day showed a decrease in small inhibitory postsynaptic current (mIPSC) frequency and an increase in the paired-pulse ratio of evoked IPSCs, suggesting a transient decrease in presynaptic GABA release in the early phase. Rats trained for 2 days showed an increase in miniature excitatory postsynaptic current (mEPSC) amplitudes/frequency and elevated AMPA/NMDA ratios, suggesting a long-term strengthening of AMPA receptor-mediated excitatory synapses. Importantly, rotor rod performance in trained rats was correlated with the mean mEPSC amplitude and the frequency obtained from that animal. In current-clamp analysis, 1-day-trained rats transiently decreased the current-induced firing rate, while 2-day-trained rats returned to pre-training levels, suggesting dynamic changes in intrinsic properties. Furthermore, western blot analysis of layer V detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in 1-day-trained rats, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in 2-day-trained rats. Finally, live-imaging analysis of Thy1-YFP transgenic mice showed that the training rapidly recruited a substantial number of spines for long-term plasticity in M1 layer V neurons. Taken together, these results indicate that motor training induces complex and diverse plasticity in M1 layer V pyramidal neurons. KEY POINTS: Here we examined motor training-induced synaptic and intrinsic plasticity of layer V pyramidal neurons in the primary motor cortex. The training reduced presynaptic GABA release in the early phase, but strengthened AMPA receptor-mediated excitatory synapses in the later phase: acquired motor performance after training correlated with the strength of excitatory synapses rather than inhibitory synapses. As to the intrinsic property, the training transiently decreased the firing rate in the early phase, but returned to pre-training levels in the later phase. Western blot analysis detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in the acute phase, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in the later phase. Live-imaging analysis of Thy1-YFP transgenic mice showed rapid and long-term spine plasticity in M1 layer V neurons, suggesting training-induced increases in self-entropy per spine.

DOI： 10.1113/JP283755

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.

DOI： 10.1021/acsami.2c03299

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

DOI： 10.1038/s41598-022-12212-6

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Herein, we discuss a new pyrene-based push-pull dye (PC) and our investigation of its photophysical properties and applicability to biological studies. The newly synthesized dye exhibits highly polarity-sensitive fluorescence over a significantly wide range (i.e., the green to far-red region), accompanied by high fluorescence quantum yields (ΦFL > 0.70 in most organic solvents) and superior photostability to that of the commonly used Nile Red (NR) dye, which also fluoresces in the green to red region. When human prostate cancer cells stained with PC were imaged using a confocal laser scanning fluorescence microscope, PC was found to selectively stain the lipid droplets. Under the cell conditions where the formation of droplets was inhibited, PC could be distributed to both the remaining droplets and the intercellular membranes, which could be distinguished based on the fluorescence solvatochromic function of PC. Furthermore, PC efficiently stained normal human skin tissue blocks treated with a transparency-enhancing agent and enabled clear visualization of individual cells in each tissue architecture by means of two-photon fluorescence microscopy (2PM). Interestingly, PC provides bright 2PM images under tissue-penetrative 960 nm excitation, realizing much clearer and deeper tissue imaging than conventional pyrene dyes and NR. These results suggest that PC could replace several commonly used dyes in various biological applications, particularly the rapid and accurate diagnosis of tissue diseases, typified by biopsy.

DOI： 10.1039/d1tb02728j

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

The use of donor-π-acceptor (D-π-A) skeletons is an effective strategy for the design of fluorophores with red-shifted emission. In particular, the use of amino and boryl moieties as the electron-donating and -accepting groups, respectively, can produce dyes that exhibit high fluorescence and solvatochromism. Herein, we introduce a dithienophosphole P-oxide scaffold as an acceptor-spacer to produce a boryl- and amino-substituted donor-acceptor-acceptor (D-A-A) π-system. The thus obtained fluorophores exhibit emission in the near-infrared (NIR) region, while maintaining high fluorescence quantum yields even in polar solvents (e.g. λ em = 704 nm and Φ F = 0.69 in CH3CN). A comparison of these compounds with their formyl- or cyano-substituted counterparts demonstrated the importance of the boryl group for generating intense emission. The differences among these electron-accepting substituents were examined in detail using theoretical calculations, which revealed the crucial role of the boryl group in lowering the nonradiative decay rate constant by decreasing the non-adiabatic coupling in the internal conversion process. The D-A-A framework was further fine-tuned to improve the photostability. One of these D-A-A dyes was successfully used in bioimaging to visualize the blood vessels of Japanese medaka larvae and mouse brain.

DOI： 10.1039/d1sc00827g

PubMed

researchmap

Language：English  

DOI： 10.1016/j.jdermsci.2021.03.004

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

High-speed two-photon microscopy can be used to analyze vascular dynamics in living animals and is essential for the understanding of brain diseases. Recent advances in fluorescent probes/optical systems have allowed successful imaging of the hippocampal vasculature in the deep brain of mice (1 mm from the brain surface) under low-speed conditions (1-2 fps); however, using high-speed techniques (>30 fps), observation of the deep-brain vasculature is still challenging. Here, a new nanoemulsion that encapsulates thousands of red-emissive pyrene dye molecules while maintaining their high two-photon brightness [1.5 x 10(2) GM (GM = 10(-50) cm(4)center dot s center dot photon(-1)center dot molecule(-1)) at 960 nm excitation] and delivers a large amount of such pyrene dyes (65 nmol) into the blood vessels of mice is developed. Remarkably, the nanoprobe is found to exploit the inherent performance of a commonly used Ti:sapphire excitation laser and a sensitive gallium arsenide phosphide nondescanned fluorescence detector to the limit, enabling visualization of the brain vasculature under the cortex region of mice (up to 1.5 mm) under very low-speed conditions. As a highlight, such a nanoprobe is successfully used to directly observe the blood flow in the hippocampal CA1 region (1.1 mm) through high-speed resonant scanning (120 fps).

DOI： 10.1002/adfm.202010698

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

To understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (magical additive glycerol improves clear alive luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MAGICAL promoted conventional confocal microscopy to visualize finer neuronal structures including synaptic boutons and spines in unprecedented deep regions, without intensive illumination leading to phototoxic effects. Fluorescence emission spectrum transmissive analysis showed that MAGICAL improved in vivo transmittance of shorter wavelength light, which is vulnerable to optical scattering, thus unsuited for in vivo microscopy. These results suggest that MAGICAL would transparentize living brains via scattering reduction.

DOI： 10.1016/j.isci.2020.101888

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.

DOI： 10.1267/ahc.20-00020

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

In vivo two-photon deep imaging with a broad field of view has revealed functional connectivity among brain regions. Here, we developed a novel observation method that utilizes a polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet with a thickness of ∼130 nm that exhibited a water retention effect and a hydrophilized adhesive surface. PEO-CYTOP nanosheets firmly adhered to brain surfaces, which suppressed bleeding from superficial veins. By taking advantage of the excellent optical properties of PEO-CYTOP nanosheets, we performed in vivo deep imaging in mouse brains at high resolution. Moreover, PEO-CYTOP nanosheets enabled to prepare large cranial windows, achieving in vivo imaging of neural structure and Ca2+ elevation in a large field of view. Furthermore, the PEO-CYTOP nanosheets functioned as a sealing material, even after the removal of the dura. These results indicate that this method would be suitable for the investigation of neural functions that are composed of interactions among multiple regions.

DOI： 10.1016/j.isci.2020.101579

PubMed

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publisher：(一社)日本癌学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

© 2020, The Author(s). The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

DOI： 10.1038/s41598-020-61579-x

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.

DOI： 10.1371/journal.pone.0237230

PubMed

researchmap

Language：English  

In order to achieve deep tissue imaging, a number of optical clearing agents have been developed. However, in a conventional microscopy setup, an objective lens can only be moved until it is in contact with a coverslip, which restricts the maximum focusing depth into a cleared tissue specimen. Until now, it is still a fact that the working distance of a high magnification objective lens with a high numerical aperture is always about 100 μm. In this study, a polymer thin film (also called as nanosheet) composed of fluoropolymer with a thickness of 130 nm, less than one-thousandth that of a 170 μm thick coverslip, is employed to replace the coverslip. Owing to its excellent characteristics, such as high optical transparency, mechanical robustness, chemical resistance, and water retention ability, nanosheet is uniquely capable of providing a coverslip-free imaging. By wrapping the tissue specimen with a nanosheet, an extra distance of 170 μm for the movement of objective lens is obtained. Results show an equivalently high resolution imaging can be obtained if a homogenous refractive index between immersion liquid and mounting media is adjusted. This method will facilitate a variety of imaging tasks with off-the-shelf high magnification objectives.

DOI： 10.1371/journal.pone.0227650

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

DOI： 10.1016/j.cell.2019.04.007

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

DOI： 10.1038/s41598-018-35412-5

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Various fluorescence microscopy techniques require bright NIR-emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus-substituted rhodamine dyes (PORs) upon substitution at the 9-position with a 2,6-dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long-term and three-color imaging in the vis-NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye-labeled biomolecule by single-molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR-emitting PREX 710-dextran conjugate.

DOI： 10.1002/anie.201804731

Web of Science

PubMed

researchmap

DOI： 10.1002/cne.24521

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OSA - The Optical Society  

We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 μm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.

DOI： 10.1364/BOE.9.002671

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

Three-dimensional (3D) super-resolution microscopy technique structured illumination microscopy (SIM) imaging of dendritic spines along the dendrite has not been previously performed in fixed tissues, mainly due to deterioration of the stripe pattern of the excitation laser induced by light scattering and optical aberrations. To address this issue and solve these optical problems, we applied a novel clearing reagent, LUCID, to fixed brains. In SIM imaging, the penetration depth and the spatial resolution were improved in LUCID-treated slices, and 160-nm spatial resolution was obtained in a large portion of the imaging volume on a single apical dendrite. Furthermore, in a morphological analysis of spine heads of layer V pyramidal neurons (L5PNs) in the medial prefrontal cortex (mPFC) of chronic dexamethasone (Dex)-treated mice, SIM imaging revealed an altered distribution of spine forms that could not be detected by high-NA confocal imaging. Thus, super-resolution SIM imaging represents a promising high-throughput method for revealing spine morphologies in single dendrites.

DOI： 10.1111/ejn.13901

Web of Science

Scopus

PubMed

researchmap

Language：English   Publisher：Blackwell Publishing Ltd  

In vivo optical imaging using fluorescence and bioluminescence is superior to other methods in terms of spatiotemporal resolution and specificity, and represents a new technology for comprehensively studying living organisms in a less invasive way. Nowadays, it is an indispensable technology for studying many aspects of cancer biology, including dynamic invasion and metastasis. In observations of fluorescence or bioluminescence signals in a living body, various problems were caused by optical characteristics such as absorption and scattering and, therefore, observation of deep tissue was difficult. Recent developments in techniques for observation of the deep tissues of living animals overcame this difficulty by improving bioluminescent proteins, fluorescent proteins, and fluorescent dyes, as well as detection technologies such as two-photon excitation microscopy. In the present review, we introduce these technological developments and in vivo application of bioluminescence and fluorescence imaging, and discuss future perspectives on the use of in vivo optical imaging technology in cancer research.

DOI： 10.1111/cas.13544

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE-INT SOC OPTICAL ENGINEERING  

In vivo two-photon microscopy is an advantageous technique for observing living mouse brains at high spatial resolutions. We previously used a 1064 nm high-power light source based on an electrically controllable gain-switched laser diode (maximum power: 4 W, repetition rate: 10 MHz, pulse width: 7.5 picoseconds) and successfully visualized EYFP expressing neurons at deeper regions in H-line mouse brains under living conditions. However, severe damages were frequently observed when the laser power after the objective lens was over 600 mW, suggesting that a higher average power might not be suitable for visualizing neural structures and functions at deep regions. To increase fluorescent signals as a strategy to avoid such invasions, here, we evaluated the effects of the excitation laser parameters such as the repetition rate (5 - 10 MHz), or the peak power, at the moderate average powers (10 - 500 mW), by taking the advantage that this electrically controllable light source could be used to change the repetition rate independently from the average power or the pulse width. The fluorescent signals of EYFP at layer V of the cerebral cortex were increased by approximately twofold when the repetition rate was decreased from 10 MHz to 5 MHz at the same average power. We also confirmed similar effects in the EYFP solution (335 mu M) and fixed brain slices. These results suggest that in vivo two-photon microscopic imaging might be improved by increasing the peak power at the same average power while avoiding the severe damages in living brains.

DOI： 10.1117/12.2288664

Web of Science

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE  

In vivo two-photon microscopy is an advantageous technique for observing living mouse brains at high spatial resolutions. We previously used a 1064 nm high-power light source based on an electrically controllable gain-switched laser diode (maximum power: 4 W, repetition rate: 10 MHz, pulse width: 7.5 picoseconds) and successfully visualized EYFP expressing neurons at deeper regions in H-line mouse brains under living conditions. However, severe damages were frequently observed when the laser power after the objective lens was over 600 mW, suggesting that a higher average power might not be suitable for visualizing neural structures and functions at deep regions. To increase fluorescent signals as a strategy to avoid such invasions, here, we evaluated the effects of the excitation laser parameters such as the repetition rate (5-10 MHz), or the peak power, at the moderate average powers (10-500 mW), by taking the advantage that this electrically controllable light source could be used to change the repetition rate independently from the average power or the pulse width. The fluorescent signals of EYFP at layer V of the cerebral cortex were increased by approximately twofold when the repetition rate was decreased from 10 MHz to 5 MHz at the same average power. We also confirmed similar effects in the EYFP solution (335 μM) and fixed brain slices. These results suggest that in vivo two-photon microscopic imaging might be improved by increasing the peak power at the same average power while avoiding the severe damages in living brains.

DOI： 10.1117/12.2288664

Scopus

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

In the field of biological microscopy technology, it is still a practical challenge to obtain high-quality tissue images, due to the tissue desiccation that occurs during observations without an effective sample mounting. Inspired by the use of plastic food wrap, this study proposes the use of polymer thin films (also known as nanosheets) to fix the tissue samples. Water-repellent nanosheets composed of the amorphous fluoropolymer CYTOP are prepared with adjustable thicknesses and their hydrophobicity, transparency, and adhesion strength are evaluated. They show excellent water-retention effect and work well for sample fixation. By wrapping cleared mouse brain slices with a 133 nm thick CYTOP nanosheet, this study achieves high spatial resolution neuron images while scanning over a large area for a long period of time. No visible artifacts arising from sample shrinkage can be detected. This study also expects that nanosheet wrapping could be effective over a longer time span by combination with conventional agarose embedding.

DOI： 10.1002/adma.201703139

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：日本バイオイメージング学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

DOI： 10.1083/jcb.201604030

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.

DOI： 10.2116/analsci.31.307

Web of Science

PubMed

researchmap

Language：English   Publisher：OXFORD UNIV PRESS  

Two-photon excitation fluorescence microscopy has become widely used in various life science fields in this decade. In the field of neuroscience in particular, in vivo two-photon microscopy has provided vital information on neural activity and brain function. In the current era of connectomics, visualization of the morphology and activity of numerous neurons in ever larger regions of the living brain are required within short periods. Based on this viewpoint, we discuss the fundamentals, advantages and potential of two-photon excitation fluorescence microscopy for the investigation of neural circuit functions.

DOI： 10.1093/jmicro/dfu110

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-mu m-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

DOI： 10.1371/journal.pone.0116280

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OPTICAL SOC AMER  

In vivo two-photon microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a two-photon microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed two-photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our two-photon microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena. (C) 2015 Optical Society of America

DOI： 10.1364/BOE.6.000891

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Recent advances in microscopy enable the acquisition of large numbers of tomographic images from living tissues. Three-dimensional microscope images are often displayed with volume rendering by adjusting the transfer functions. However, because the emissions from fluorescent materials and the optical properties based on point spread functions affect the imaging results, the intensity value can differ locally, even in the same structure. Further, images obtained from brain tissues contain a variety of neural structures such as dendrites and axons with complex crossings and overlapping linear structures. In these cases, the transfer functions previously used fail to optimize image generation, making it difficult to explore the connectivity of these tissues.
Results: This paper proposes an interactive visual exploration method by which the transfer functions are modified locally and interactively based on multidimensional features in the images. A direct editing interface is also provided to specify both the target region and structures with characteristic features, where all manual operations can be performed on the rendered image. This method is demonstrated using two-photon microscope images acquired from living mice, and is shown to be an effective method for interactive visual exploration of overlapping similar structures.
Conclusions: An interactive visualization method was introduced for local improvement of visualization by volume rendering in two-photon microscope images containing regions in which linear nerve structures crisscross in a complex manner. The proposed method is characterized by the localized multidimensional transfer function and interface where the parameters can be determined by the user to suit their particular visualization requirements.

DOI： 10.1186/s12859-014-0415-x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Background: Dendrites of cortical neurons are widely spread across several layers of the cortex. Recently developed two-photon microscopy systems are capable of visualizing the morphology of neurons within deeper layers of the brain and generate large amounts of volumetric imaging data from living tissue.
Method: For visual exploration of the three-dimensional (3D) structure of dendrites and the connectivity among neurons in the brain, we propose a visualization software and interface for 3D images based on a new transfer function design using volume rendered feature spaces. This software enables the visualization of multidimensional descriptors of shape and texture extracted from imaging data to characterize tissue. It also allows the efficient analysis and visualization of large data sets.
Results: We apply and demonstrate the software to two-photon microscopy images of a living mouse brain. By applying the developed visualization software and algorithms to two-photon microscope images of the mouse brain, we identified a set of feature values that distinguish characteristic structures such as soma, dendrites and apical dendrites in mouse brain. Also, the visualization interface was compared to conventional 1D/2D transfer function system.
Conclusions: We have developed a visualization tool and interface that can represent 3D feature values as textures and shapes. This visualization system allows the analysis and characterization of the higher-dimensional feature values of living tissues at the micron level and will contribute to new discoveries in basic biology and clinical medicine. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.compbiomed.2014.07.007

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OPTICAL SOC AMER  

In this study, we investigated the picosecond optical pulse generation from a 1064-nm distributed feedback laser diode under strong gain switching. The spectrum of the generated optical pulses was manipulated in two different ways: (i) by extracting the short-wavelength components of the optical pulse spectrum and (ii) by compensating for spectral chirping in the extracted mid-spectral region. Both of these methods shortened the optical pulse duration to approximately 7 ps. These optical pulses were amplified to over 20-kW peak power for two-photon microscopy. We obtained clear two-photon images of neurons in a fixed brain slice of H-line mouse expressing enhanced yellow fluorescent protein. Furthermore, a successful experiment was also confirmed for in vivo deep region H-line mouse brain neuron imaging. (C) 2014 Optical Society of America

DOI： 10.1364/OE.22.005746

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

In vivo two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power, and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.

DOI： 10.1038/srep01014

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Left-right (L-R) asymmetry is a fundamental feature of higher-order neural function. However, the molecular basis of brain asymmetry remains unclear. We recently reported L-R asymmetry of hippocampal circuitry caused by differential allocation of N-methyl-D-aspartate receptor (NMDAR) subunit GluR epsilon 2 (NR2B) in hippocampal synapses. Using electrophysiology and immunocytochemistry, here we analyzed the hippocampal circuitry of the inversus viscerum (iv) mouse that has a randomized laterality of internal organs. The iv mouse hippocampus lacks L-R asymmetry, it exhibits right isomerism in the synaptic distribution of the epsilon 2 subunit, irrespective of the laterality of visceral organs. This independent right isomerism of the hippocampus is the first evidence that a distinct mechanism downstream of the iv mutation generates brain asymmetry.

DOI： 10.1371/journal.pone.0001945

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Input-dependent left-right asymmetry of NMDA receptor epsilon 2 (NR2B) subunit allocation was discovered in hippocampal Schaffer collateral (Sch) and commissural fiber pyramidal cell synapses (Kawakami et al., 2003). To investigate whether this asymmetrical epsilon 2 allocation is also related to the types of the postsynaptic cells, we compared postembedding immunogold labeling for epsilon 2 in left and right Sch synapses on pyramidal cells and interneurons. To facilitate the detection of epsilon 2 density difference, we used epsilon 1 (NR2A) knock-out (K0) mice, which have a simplified NMDA receptor subunit composition.
The labeling density for epsilon 2 but not zeta 1 (NR1) and subtype 2/3 glutamate receptor (GluR2/3) in Sch-CA1 pyramidal cell synapses was significantly different between the left and right hippocampus with opposite directions in strata oriens and radiatum; the left to right ratio of epsilon 2 labeling density was 1:1.50 in stratum oriens and 1.44:1 in stratum radiatum. Nosignificant difference, however, was detected in CA1 stratum radiatum between the left and right Sch-GluR4-positive (mostly parvalbumin-positive) and Sch-GluR4-negative interneuron synapses. Consistent with the anatomical asymmetry, the amplitude ratio of NMDA EPSCs to non-NMDA EPSCs in pyramidal cells was approximately two times larger in right than left stratum radiatum and vice versa in stratum oriens of epsilon 1 K0 mice. Moreover, the amplitude of long-term potentiation in the Sch-CA1 synapses of left stratum radiatum was significantly larger than that in the right corresponding synapses. These results indicate that the asymmetry of epsilon 2 distribution is target cell specific, resulting in the left-right difference in NMDA receptor content and plasticity in Sch-CA1 pyramidal cell synapses in epsilon 1 K0 mice.

DOI： 10.1523/JNEUROSCI.2134-05.2005

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Despite its implications for higher order functions of the brain, little is currently known about the molecular basis of left-right asymmetry of the brain. Here we report that synaptic distribution of N-methyl-D-aspartate (NMDA) receptor GluRepsilon2 (NR2B) subunits in the adult mouse hippocampus is asymmetrical between the left and right and between the apical and basal dendrites of single neurons. These asymmetrical allocations of epsilon2 subunits differentiate the properties of NMDA receptors and synaptic plasticity between the left and right hippocampus. These results provide a molecular basis for the structural and functional asymmetry of the mature brain.

DOI： 10.1126/science.1082609

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Hippocampal CA3 pyramidal neurons receive synaptic inputs from commissural and associational fibers on both apical and basal dendrites. NMDA receptors at these synapses were examined in hippocampal slices of wild-type mice and GluR epsilon 1 (NR2A) subunit knockout mice. Electrical stimulations at the CA3 stratum radiatum or stratum oriens activate both commissural and associational (C/A).synapses, whereas stimulations at ventral fimbria mainly activate commissural synapses. Ro 25-6981 and ifenprodil, the GluR epsilon 2 (NR2B) subunit-selective NMDA receptor antagonists, suppressed NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) at the commissural-CA3 synapses on basal dendrites more strongly than those at the C/A-CA3 synapses on apical or basal dendrites. However, glutamate-evoked NMDA receptor currents were reduced by the GluR epsilon 1 subunit knockout to a similar extent at both apical and basal dendrites. The GluR epsilon 1 subunit knockout also reduced NMDA EPSCs at the C/A-CA3 synapses on basal dendrites, but did not affect NMDA EPSCs at the commissural-CA3 synapses on basal dendrites. These results confirmed our previous findings that NMDA receptors operating at different synapses in CA3 pyramidal cells have different GluR epsilon subunit compositions, and further show that the GluR epsilon subunit composition may be regulated depending on the types of synaptic inputs, even within a single CA3 pyramidal neuron. (C) 2000 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0028-3908(99)00217-8

Web of Science

PubMed

researchmap

Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)  

CiNii Books

researchmap

Language：Japanese  

DOI： 10.11477/mf.1408201063

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公財)金原一郎記念医学医療振興財団  

生体組織標本の深部イメージングを可能にする多光子顕微鏡技術は,神経科学の分野を中心に,生物学,医学研究への応用が広がりつつある。本稿では,近年の筆者らの研究グループにおける取り組み例を示しながら,in vivo 2光子顕微鏡観察の新たな展開について紹介する。(著者抄録)

researchmap

Language：Japanese  

CiNii Books

J-GLOBAL

researchmap

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

J-GLOBAL

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本比較内分泌学会  

DOI： 10.5983/nl2008jsce.41.136

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2016065694

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

J-GLOBAL

researchmap

Language：Japanese   Publisher：The Japanese Society of Plant Morphology  

For the field of neuroscience, laser-scanning florescence microscopy utilizing two-(or multi-) photon excitation process (two-(multi-)photon excitation laser scanning microscopy, two-(multi-)photon microscopy) has become widely used as an essential tool for biological and medical research including cancer, and immune researches. Especially, "<i>in vivo</i>" two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, that enabled us to identify individual fluorescent beads of which diameter is 170 nm; smaller than classical PSF width. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.

DOI： 10.5685/plmorphol.26.31

CiNii Books

researchmap

Language：Japanese  

CiNii Books

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本生物物理学会  

DOI： 10.2142/biophys.54.035

CiNii Books

J-GLOBAL

researchmap

Language：Japanese  

DOI： 10.11477/mf.2425101554

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

researchmap

Language：Japanese   Publisher：レーザー学会  

CiNii Books

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：エヌ・ティー・エス  

CiNii Books

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

researchmap

Language：Japanese   Publisher：公益社団法人 日本薬学会  

DOI： 10.14894/faruawpsj.47.8_724

CiNii Books

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2011.07.773

Web of Science

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.711

Web of Science

researchmap