非破壊に生体内ヘムタンパク質の酸化還元状態を分析する線形・非線形顕微分光法の開発

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 小澤 岳昌, 東京大学教授 塩谷 光彦, 東京大学教授 長谷川 哲也, 東京大学准教授 狩野 直和, 東京大学准教授 岡林 潤University of Tokyo(東京大学

Place & Play SERS: sample collection and preparation-free surface-enhanced Raman spectroscopy

The ability to perform sensitive, real-time, in situ, multiplex chemical analysis is indispensable for diverse applications such as human health monitoring, food safety testing, forensic analysis, environmental sensing, and homeland security. Surface-enhanced Raman spectroscopy (SERS) is an effective tool to offer the ability by virtue of its high sensitivity and rapid label-free signal detection as well as the availability of portable Raman spectrometers. Unfortunately, the practical utility of SERS is limited because it generally requires sample collection and preparation, namely, collecting a sample from an object of interest and placing the sample on top of a SERS substrate to perform a SERS measurement. In fact, not all analytes can satisfy this requirement because the sample collection and preparation process may be undesirable, laborious, difficult, dangerous, costly, or time-consuming. Here we introduce "Place & Play SERS" based on an ultrathin, flexible, stretchable, adhesive, biointegratable gold-deposited polyvinyl alcohol (PVA) nanomesh substrate that enables placing the substrate on top of an object of interest and performing a SERS measurement of the object by epi-excitation without the need for touching, destroying, and sampling it. Specifically, we characterized the sensitivity of the gold/PVA nanomesh substrate in the Place & Play SERS measurement scheme and then used the scheme to conduct SERS measurements of both wet and dry objects under nearly real-world conditions. To show the practical utility of Place & Play SERS, we demonstrated two practical applications: food safety testing and forensic analysis. Our results firmly verified the new measurement scheme of SERS and are expected to extend the potential of SERS by opening up untapped applications of sensitive, real-time, in situ multiplex chemical analysis.Comment: 11 pages, 7 figure

Identification of intracellular squalene in living algae, Aurantiochytrium mangrovei with hyper-spectral coherent anti-Stokes Raman microscopy using a sub-nanosecond supercontinuum laser source

We applied hyper-spectral coherent anti-Stokes Raman scattering imaging to intracellular lipid identification in living microalgae, Aurantiochytrium mangrovei 18W-13a. Two different lipids, squalene and triacylglycerol, were found inside living cells with clear vibrational contrast. Based on the endogenous lipid band as a result of the cis C[DOUBLE BOND]C stretch vibrational mode, squalene and triacylglycerol were clearly distinguished in different intracellular areas. In particular, squalene was detected solely in vacuoles as lipid particles, which was also supported by electron microscopy

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms

Transport of carbon-bearing dusts from Iraq to Japan during Iraq\u27s War

Rainwater collected during the period of February-April, 2003 in Kanazawa University and Hakui, Ishikawa Prefecture, Japan, was investigated by ion chromatography of the dissolved ions, and Scanning electron microscopy equipped with energy dispersive analyzer (SEM-EDX), electron microprobe analyzer (EPMA), transmission electron microscopy, and X-ray powder diffraction (XRD) analyses of the filtered samples. The pH, Eh, EC, dissolved oxygen (DO) and depleted U (DU) of the rainwater were also measured. The results show a rare case with low pH value of rain and high value of electrical conductivity (EC) in the rainwater during certain period. The NO 3 -, SO 4 2- and NH 4 + concentrations on 26th March, show quite high values after oil field fires on 21st March 2003. The powdery dust and carbon-bearing nm sized particles contained in the rainwater have characteristics different from that of the Asian dust (Kosa). Since 21 March, 2003 the Iraq plunged into the war, the 9 oil field fires were reported on 25 March, 2003 in Rumaylah oil field near the borderline of Kuwait. The first precipitation was caught in Kanazawa on 24 March, 2003. The pH value of the rainwater indicated a strong acid rain (pH 3.6) with black powdery dusts. The acid rain on 27th March to 2nd April (pH 3.4-4.3; 5-17mm) associated with WNW wind (2.7-4.1m/s) has continued to the first 10 days of April in Kanazawa, during the 9 oil fields were burning in Iraq. The EC in rainwater collected from 24 March to 15 April, 2003, shows high concentrations of ions, suggesting high contents of water soluble-carbon particles and soot. In the meantime, it was the highest season of sandstorms at bare dried land in Iraq from the end of March to the beginning of April. Westerly wind blew around 5500m high, and the sandstorm with 2000m wide was transported by strong wind to Zagros 3000m high mountains. The hot wind hit the Zagros Mountains and easily to join and rise to Westerly wind high by a rising air current. The 234U/ 238U activity (around 0.2) in typical depleted uranium (DU) is quite low compared with that (around 1.0) in natural uranium. The contamination of DU from the Iraq conflict may be, if any, negligibly small on undetectable level. On the other hand, the powdery dusts and carbon-bearing particles of rainwater have been produced by combustion of oil field in Iraq and they are directly cycling in our planet by Westerly wind. Black carbon particles are a product of incomplete combustion, and are the principal light-absorbing atmospheric aerosol. The result reminds us of the scientific values of short-term atmospheric environment records during Iraq\u27s War. This has consequences for primary production of powdery dusts coming from Iraq to the atmosphere over half the globe away. © 2004 Elsevier Ltd. All rights reserved

Linear and nonlinear Raman microspectroscopy: History, instrumentation, and applications

International audienceThrough carrying out Raman spectroscopy under a microscope, we can observe the molecular distribution and dynamics of living cells in situ without staining or labeling. In general, Raman microscopy/microspectroscopy is classified into spontaneous Raman microscopy/microspectroscopy and nonlinear Raman microscopy/microspectroscopy. This review will first summarize the historical development of Raman microscopes, and will discuss in detail their instrumentation and applications

Hyperspectral coherent Raman imaging – principle, theory, instrumentation, and applications to life sciences

International audienceCoherent Raman scattering microscopy such as coherent anti-Stokes Raman scattering and stimulated Raman scattering microscopy boosts the weak Raman signal and enables us to perform label-free visualization of the molecular distribution and its dynamical behavior in living cells and tissues with high speed. In comparison with fluorescence imaging, cells and tissues can be visualized without specifying the target molecule. In this review, we describe the characteristics of a hyperspectral coherent Raman imaging method, which is capable of acquiring both spectra and images simultaneously, and review applications to life sciences. Copyright © 2015 John Wiley & Sons, Ltd

Half-substituted fluorocycloparaphenylenes with high symmetry: Synthesis, properties and derivatization to densely substituted carbon nanorings

Cycloparaphenylenes (CPPs) are ring-shaped aromatic hydrocarbons in which benzene rings are connected to each other at the para-positions. In recent years, fluorinated CPPs (FCPPs) have attracted much attention as electron-accepting CPPs as well as strained fluoroarenes. Herein, we report the synthesis, properties, and derivatization of novel FCPPs; F16[8]CPP (1) and F12[6]CPP (2), in which ortho-difluorophenylene units are circularly connected to form CPP structure. The short-step synthesis of 1 and 2 has been achieved by the strategy using macrocyclic nickel or gold complexes. Furthermore, the derivatization of 1 was successfully achieved to afford a new heteroring-containing carbon nanoring; hexadecapyrrolyl[8]cycloparaphenylene 5, where 16 pyrrole rings are densely substituted on CPP framework

Multimodal Imaging of Living Cells with Multiplex Coherent Anti-stokes Raman Scattering (CARS), Third-order Sum Frequency Generation (TSFG) and Two-photon Excitation Fluorescence (TPEF) Using a Nanosecond White-light Laser Source

International audienceThe subnanosecond "white-light laser" source has been applied to multimodal, multiphoton, and multiplex spectroscopic imaging (M(3) spectroscopic imaging) with coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and two-photon excitation fluorescence (TPEF). As the proof-of-principle experiment, we performed simultaneous imaging of polystyrene beads with TSFG and TPEF. This technique is then applied to live cell imaging. Mouse L929 fibroblastic cells are clearly visualized by CARS, TSFG, and TPEF processes. M(3) spectroscopic imaging provides various and unique cellular information with different image contrast based on each multiphoton process

Electronically resonant third-order sum frequency generation spectroscopy using a nanosecond white-light supercontinuum

International audienceThird-order sum frequency generation (TSFG) is one of the third-order nonlinear optical processes, and has the generation mechanism analogous to third harmonic generation (THG). By using a white-light supercontinuum, we can obtain broadband multiplex TSFG spectra. In the present study, we developed an electronically resonant TSFG spectrometer, and applied it to obtain TSFG spectra of hemoproteins. Analyzed TSFG ratio spectra clearly showed the resonant enhancement attributable to the electronic state of hemoproteins. This is a promising method for the imaging of electronic states of molecules inside living cells or tissues