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The surface plasmon resonance (SPR) technique has been widely applied to biosensing technologies for the rapid quantification of biomolecules without enzyme and fluorescent labeling. However, the conventional prism-coupling SPR method generally has a detection area of a few mm2, and the large contribution of the background signal forms a barrier to highly sensitive detection. Based on a highly spatially resolved SPR method, the present study constructed a scanning GC-SPR imaging instrument using an objective lens with a high numerical aperture and a plasmonic chip that could be used for grating-coupled SPR. Focusing light on the diffraction limit can suppress background signals and improve detection sensitivity. SPR imaging can also be performed by scanning a focal spot. Using this method, the refractive index of a mixture of water and dimethyl sulfoxide was measured with a detection accuracy of 2.43 × 10-3 RIU. Polydopamine films prepared with a thickness of <5 nm were also measured, and each film thickness was evaluated with high sensitivity from the effective refractive index detected in a small area of <1 µm2.
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To promote the clinical application of human induced pluripotent stem cell (hiPSC)-derived hepatocytes, a method capable of monitoring regenerative processes and assessing differentiation efficiency without harming or modifying these cells is important. Raman microscopy provides a powerful tool for this as it enables label-free identification of intracellular biomolecules in live samples. Here, we used label-free Raman microscopy to assess hiPSC differentiation into hepatocyte lineage based on the intracellular chemical content. We contrasted these data with similar phenotypes from the HepaRG and from commercially available hiPSC-derived hepatocytes (iCell hepatocytes). We detected hepatic cytochromes, lipids, and glycogen in hiPSC-derived hepatocyte-like cells (HLCs) but not biliary-like cells (BLCs), indicating intrinsic differences in biomolecular content between these phenotypes. The data show significant glycogen and lipid accumulation as early as the definitive endoderm transition. Additionally, we explored the use of Raman imaging as a hepatotoxicity assay for the HepaRG and iCell hepatocytes, with data displaying a dose-dependent reduction of glycogen accumulation in response to acetaminophen. These findings show that the nondestructive and high-content nature of Raman imaging provides a promising tool for both quality control of hiPSC-derived hepatocytes and hepatotoxicity screening.
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Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Pluripotentes Induzidas , Humanos , Hepatócitos , Diferenciação CelularRESUMO
Simultaneous observation of drug distribution at the effector site and subsequent cell response are essential in the drug development process. However, few studies have visualized the drug itself and biomolecular interactions in living cells. Here, we used label-free Raman microscopy to investigate drug-induced cytotoxicity and visualize drug uptake and subcellular localization by its specific molecular fingerprint. A redox-sensitive Raman microscope detected the decrease of reduced cytochrome c (cyt c) after Actinomycin D (ActD) treatment in a time-dependent and dose-dependent format. Immunofluorescence staining of cyt c suggested that the release of cyt c was not the major cause. Combining Raman microscopy with conventional biological methods, we reported that the oxidization of cyt c is an early cytotoxicity marker prior to the release of cyt c. Moreover, as the spectral properties of ActD are sensitive to the surrounding environment, subcellular localization of ActD was visualized sensitively by the weak autofluorescence, and the intercalation of ActD into DNA was detected by shifted Raman peaks, allowing for parallel observation of drug uptake and the mechanism of action. In this research, we achieved simultaneous observation of cytotoxicity and cellular drug uptake by Raman microscopy, which could facilitate a precise understanding of pharmacological effects and predict potential drug toxicity in the future.
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Citocromos c , Análise Espectral Raman , Dactinomicina/farmacologia , Microscopia , Imagem Óptica , Análise Espectral Raman/métodosRESUMO
We demonstrated resolution improvement in two-photon excitation microscopy by combining saturated excitation (SAX) of fluorescence and pupil manipulation. We theoretically estimated the resolution improvement and the sidelobe effect in the point spread function with various pupil designs and found that the combination of SAX and core-ring illumination can effectively enhance the spatial resolution in 3D and suppress sidelobe artifacts. The experimental demonstration shows that the proposed technique is effective for observation with a depth of 100 µm in a tissue phantom and can be applied to 3D observations of tissue samples with higher spatial resolution than conventional two-photon excitation microscopy.
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Iluminação , Microscopia de Fluorescência/métodos , Fótons , Corantes Fluorescentes/química , Células HeLa , Humanos , Imagens de FantasmasRESUMO
High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.
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Elétrons , Microscopia/métodos , Fenômenos Ópticos , Osteoblastos/citologia , Coloração e Rotulagem , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Sobrevivência Celular , Imageamento Tridimensional , Camundongos , Mitocôndrias/ultraestrutura , Osteoblastos/ultraestruturaRESUMO
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.
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Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.
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Cor , Elétrons , Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Nanodiamantes/análise , Nanodiamantes/química , Sobrevivência Celular , Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Fluorescência/instrumentaçãoRESUMO
We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.
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Movimento Celular/fisiologia , Rastreamento de Células/métodos , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Elétrons , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Although investigating drug modulation of cytochrome P450 (CYP) activity under physiological conditions is crucial in drug development to avoid severe adverse drug reactions, the current evaluation approaches that rely on the destructive and end-point analysis can be misleading due to invasive treatments and cellular heterogeneity. Here, we propose a non-destructive and high-content method for visualizing and quantifying intracellular CYP activity under drug administration by Raman microscopy. The redox-state and spin-state sensitive Raman measurement indicated that the induced CYPs in living hepatocytes were in oxidized and low-spin state, which is related to monooxygenase function of CYP. Moreover, glycogen depletion associated with CYP induction was simultaneously observed, indicating a relevant effect on glucose metabolism. By deciphering the overall changes in the biochemical fingerprints of hepatocytes, Raman microscopy offers a non-destructive and quantitative chemical imaging method to evaluate CYP activity at the single-cell level with the potential to facilitate future drug development schemes.
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Sistema Enzimático do Citocromo P-450 , Microscopia , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática , Hepatócitos/metabolismoRESUMO
The transmission of neuronal information is propagated through synapses by neurotransmitters released from presynapses to postsynapses. Neurotransmitters released from the presynaptic vesicles activate receptors on the postsynaptic membrane. Glutamate acts as a major excitatory neurotransmitter for synaptic vesicles in the central nervous system. Determining the concentration of glutamate in single synaptic vesicles is essential for understanding the mechanisms of neuronal activation by glutamate in normal brain functions as well as in neurological diseases. However, it is difficult to detect and quantitatively measure the concentration of glutamate in single synaptic vesicles owing to their small size, i.e., â¼40 nm. In this study, to quantitatively evaluate the concentrations of the contents in small membrane-bound vesicles, we developed an optical trapping Raman spectroscopic system that analyzes the Raman spectra of small objects captured using optical trapping. Using artificial liposomes encapsulating glutamate that mimic synaptic vesicles, we investigated whether spontaneous Raman scattered light of glutamate can be detected from vesicles trapped at the focus using optical forces. A 575 nm laser beam was used to simultaneously perform the optical trapping of liposomes and the detection of the spontaneous Raman scattered light. The intensity of Raman scattered light that corresponds to lipid bilayers increased with time. This observation suggested that the number of liposomes increased at the focal point. The number of glutamate molecules in the trapped liposomes was estimated from the calibration curve of the Raman spectra of glutamate solutions with known concentration. This method can be used to measure the number of glutamate molecules encapsulated in synaptic vesicles in situ.
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The excitatory synaptic transmission is mediated by glutamate in neuronal networks of the mammalian brain. In addition to the synaptic glutamate, extra-synaptic glutamate is known to modulate the neuronal activity. In neuronal networks, glutamate uptake is an important role of neurons and glial cells for lowering the concentration of extracellular glutamate and to avoid the excitotoxicity by glutamate. Monitoring the spatial distribution of intracellular glutamate is important to study the uptake of glutamate, but the approach has been hampered by the absence of appropriate glutamate analogs that report the localization of glutamate. Deuterium-labeled glutamate (GLU-D) is a promising tracer for monitoring the intracellular concentration of glutamate, but physiological properties of GLU-D have not been studied. Here we study the effects of extracellular GLU-D for the neuronal activity by using primary cultured rat hippocampal neurons that form neuronal networks on microelectrodes array. The frequency of firing in the spontaneous activity of neurons increased with the increasing concentration of extracellular GLU-D. The frequency of synchronized burst activity in neurons increased similarly as we observed in the spontaneous activity. These changes of the neuronal activity with extracellular GLU-D were suppressed by antagonists of glutamate receptors. These results suggest that GLU-D can be used as an analog of glutamate with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of glutamate for studying the dynamics of glutamate during neuronal activity.
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We present a technique for improving the spatial resolution of two-photon excitation microscopy; our technique combines annular illumination with an in situ estimation of the point spread function (PSF) used for deconvolution. For the in situ estimation of the PSF, we developed a technique called autocorrelation scanning, in which a sample is imaged by the scanning of two excitation foci that are overlapped over various distances. The image series obtained with the variation of the distance between the two foci provides the autocorrelation function of the PSF, which can be used to estimate the PSF at specific positions within a sample. We proved the principle and the effectiveness of this technique through observations of a fluorescent biological sample, and we confirmed that the improvement in the spatial resolution was ~1.7 times that of typical two-photon excitation microscopy by observing a mouse brain phantom at a depth of 200 µm.
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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.
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Microscopia , Imagem Óptica , Estimulação Luminosa , Animais , Dineínas do Axonema/ultraestrutura , Células CHO , Cricetulus , Desenho de Equipamento , Ouro , Nanopartículas Metálicas , Microscopia/instrumentação , Imagem Óptica/instrumentação , Estimulação Luminosa/instrumentação , Receptores Imunológicos/metabolismo , Óxido de ZincoRESUMO
Intracellular structures of HeLa cells are observed using a direct electron beam excitation-assisted fluorescence (D-EXA) microscope. In this microscope, a silicon nitride membrane is used as a culture plate, which typically has a low biocompatibility between the sample and the silicon nitride surface to prevent the HeLa cells from adhering strongly to the surface. In this work, the surface of silicon nitride is modified to allow strong cell attachment, which enables high-resolution observation of intracellular structures and an increased signal-to-noise ratio. In addition, the penetration depth of the electron beam is evaluated using Monte Carlo simulations. We can conclude from the results of the observations and simulations that the surface modification technique is promising for the observation of intracellular structures using the D-EXA microscope.
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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.
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Microscopia de Contraste de Fase , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Elétrons , Ouro/química , Células HeLa , Humanos , Nanopartículas Metálicas/química , Tamanho da Partícula , Razão Sinal-Ruído , Silicatos/química , Imagem com Lapso de Tempo , Compostos de Zinco/químicaRESUMO
We have developed electron beam excitation assisted (EXA) optical microscope[1-3], and demonstrated its resolution higher than 50 nm. In the microscope, a light source in a few nanometers size is excited by focused electron beam in a luminescent film. The microscope makes it possible to observe dynamic behavior of living biological specimens in various surroundings, such as air or liquids. Scan speed of the nanometric light source is faster than that in conventional near-field scanning optical microscopes. The microscope enables to observe optical constants such as absorption, refractive index, polarization, and their dynamic behavior on a nanometric scale. The microscope opens new microscopy applications in nano-technology and nano-science.Figure 1(a) shows schematic diagram of the proposed EXA microscope. An electron beam is focused on a luminescent film. A specimen is put on the luminescent film directly. The inset in Fig. 1(a) shows magnified image of the luminescent film and the specimen. Nanometric light source is excited in the luminescent film by the focused electron beam. The nanometric light source illuminates the specimen, and the scattered or transmitted radiation is detected with a photomultiplier tube (PMT). The light source is scanned by scanning of the focused electron beam in order to construct on image. Figure 1(b) shows a luminescence image of the cells acquired with the EXA microscope, and Fig. 1(c) shows a phase contrast microscope image. Cells were observed in culture solution without any treatments, such as fixation and drying. The shape of each cell was clearly recognized and some bright spots were observed in cells. We believe that the bright spots indicated with arrows were auto-fluorescence of intracellular granules and light- grey regions were auto-fluorescence of cell membranes. It is clearly demonstrated that the EXA microscope is useful tool for observation of living biological cells in physiological conditions.jmicro;63/suppl_1/i16/DFU090F1F1DFU090F1Fig. 1.(a) Optical setup of EXA microscpe, and observation results of of living MARCO-expressing CHO cells with (b) EXA microscope and (c) phase contrast microscope. We proposed the EXA microscope as a technique with high spatial resolution beyond the diffraction limit of light. A spatial resolution greater than 100 nm was achieved for the EXA microscope and the dynamic behavior of moving nanoparticles in water was observed by time lapse imaging. We also demonstrated luminescence image of living cells in culture solution without any treatments.
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We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam. Electron beam excitation enables detailed observations on the nanometer scale. Real-time live-cell observation is also possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation. In this study, we demonstrated observation of cellular components by autofluorescence excited with a focused electron beam and performed dynamic observations of intracellular granules. Since autofluorescence is associated with endogenous substances in cells, this microscope can also be used to investigate the intrinsic properties of organelles.