Seiichi Uchiyama

The University of Tokyo, Tōkyō, Japan

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Publications (57)289.46 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We developed new cationic fluorescent polymeric thermometers containing both benzothiadiazole and BODIPY units as an environment-sensitive fluorophore and as a reference fluorophore, respectively. The temperature-dependent fluorescence spectra of the thermometers enabled us to perform highly sensitive and practical ratiometric temperature sensing inside living mammalian cells. Intracellular temperatures of non-adherent MOLT-4 (human acute lymphoblastic leukaemia) and adherent HEK293T (human embryonic kidney) cells could be monitored with high temperature resolutions (0.01–1.0 °C) using the new cationic fluorescent polymeric thermometer.
    The Analyst 05/2015; 140(13). DOI:10.1039/C5AN00420A · 4.11 Impact Factor
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    Teruyuki Hayashi · Nanaho Fukuda · Seiichi Uchiyama · Noriko Inada
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    ABSTRACT: Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.
    PLoS ONE 02/2015; 10(2):e0117677. DOI:10.1371/journal.pone.0117677 · 3.23 Impact Factor
  • Toshikazu Tsuji · Satoshi Yoshida · Aruto Yoshida · Seiichi Uchiyama
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    ABSTRACT: An accurate method for measuring intracellular temperature is potentially valuable because the temperature inside a cell can correlate with diverse biological reactions and functions. In a previous study, we reported the use of a fluorescent polymeric thermometer to reveal intracellular temperature distributions, but this polymer required microinjection for intracellular use, such that it was not user-friendly; furthermore, it could not be used in small cells or cells with a cell wall, such as yeast. In the present study, we developed several novel cationic fluorescent copolymers, including NN-AP2.5 and NN/NI-AP2.5, which exhibited spontaneous and rapid entry (≤ 20 min) into yeast cells and subsequent stable retention in the cytoplasm. The fluorescence lifetime of NN-AP2.5 in yeast cells was temperature-dependent (6.2 ns at 15°C and 8.6 ns at 35°C), and the evaluated temperature resolution was 0.09-0.78°C within this temperature range. In addition, NN-AP2.5 and NN/NI-AP2.5 readily entered and functioned within mammalian cells. Taken together, these data show that our novel cationic fluorescent polymeric thermometers enable accurate and practical intracellular thermometry in a wide range of cells without the need for a microinjection procedure.
    Analytical Chemistry 09/2013; 85(20). DOI:10.1021/ac402128f · 5.64 Impact Factor
  • Noriko Inada · Seiichi Uchiyama
    Imaging in medicine 08/2013; 5(4):303-305. DOI:10.2217/iim.13.34
  • Kyoko Kawamoto · Seiichi Uchiyama
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    ABSTRACT: We introduce 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzothiadiazole and 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzoselenadiazole as new fluorogenic reagents for amines. These reagents are nonfluorescent themselves and can easily react with nonfluorescent amines to produce environment-sensitive fluorophores. Herein, we report the synthesis of the new reagents and the fluorescence properties of the reagents and their amine derivatives as well as the reactivity of the new reagents toward amines.
    ChemInform 04/2013; 44(14). DOI:10.1002/chin.201314206
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    Kohki Okabe · Seiichi Uchiyama · Noriko Inada · Yoshie Harada · Takashi Funatsu
    Biophysical Journal 01/2013; 104(2):201-. DOI:10.1016/j.bpj.2012.11.1137 · 3.97 Impact Factor
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    ABSTRACT: Chemical mitochondrial uncouplers are lipophilic weak acids that increase proton transport into the mitochondrial matrix via a pathway independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. These molecules enable determination of maximal cellular respiration and have antioxidant effects that protect from ischemia-reperfusion injury. However, the most widely used proton transporter uncouplers have off-target activity that leads to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane, we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4–e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane and protects mice from acute renal ischemic-reperfusion injury. Thus, BAM15 represents a reliable new tool for the analysis of cellular bioenergetic function that has therapeutic potential by altering mitochondrial function in vivo.
    Molecular Metabolism 01/2013; 3(2). DOI:10.1016/j.molmet.2013.11.005
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    ABSTRACT: An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.
    Chemistry - A European Journal 07/2012; 18(31):9552-63. DOI:10.1002/chem.201200597 · 5.73 Impact Factor
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    ABSTRACT: A coumarin analogue, 8-methoxy-4-methyl-2H-benzo[g]chromen-2-one (MMBC), is almost non-fluorescent in non-polar media, whereas it exhibits dramatically enhanced fluorescence in polar protic solvents. This study investigates the mechanistic features of the significant solvent effects on the fluorescence properties of MMBC and a related compound, 4-methyl-2H-benzo[g]chromen-2-one (MBC), by time-resolved fluorescence and photoacoustic measurements and by theoretical calculations. Time-resolved photoacoustic measurements reveal that the extremely fast non-radiative processes of MBC and MMBC in non-polar solvents can be attributed predominantly to internal conversion. The internal conversion rates of MBC and MMBC are remarkably reduced in a rigid matrix of 3-methylpentane at 77 K, suggesting that internal conversion is a thermally activated process. The photophysical properties of MBC and MMBC examined in selected solvents with different polarities and hydrogen-bond donating abilities show that the internal conversion rate is greatly reduced by hydrogen-bonding interactions with protic solvents. Furthermore, remarkable fluorescence enhancement is observed by adding a small amount of trifluoroethanol to n-hexane solutions of MBC and MMBC, indicating that internal conversion is suppressed by formation of hydrogen-bonded complexes with protic solvents. In light of theoretical considerations based on time-dependent density functional theory (TD-DFT) and INDO/S-CI calculations, the occurrence of fast internal conversion in MBC and MMBC can be explained in terms of the proximity effect, i.e., pseudo Jahn-Teller coupling between energetically close S(1) and S(2) states.
    Photochemical and Photobiological Sciences 06/2012; 11(8):1368-76. DOI:10.1039/c2pp25055a · 2.27 Impact Factor
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    ABSTRACT: Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.
    Nature Communications 02/2012; 3(1):705. DOI:10.1038/ncomms1714 · 11.47 Impact Factor
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    Kohki Okabe · Seiichi Uchiyama · Yoshie Harada · Takashi Funatsu
    Biophysical Journal 01/2012; 102(3):198-. DOI:10.1016/j.bpj.2011.11.1078 · 3.97 Impact Factor
  • A Prasanna de Silva · Seiichi Uchiyama
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    ABSTRACT: The competition between Photoinduced electron transfer (PET) and other de-excitation pathways such as fluorescence and phosphorescence can be controlled within designed molecular structures. Depending on the particular design, the resulting optical output is thus a function of various inputs such as ion concentration and excitation light dose. Once digitized into binary code, these input-output patterns can be interpreted according to Boolean logic. The single-input logic types of YES and NOT cover simple sensors and the double- (or higher-) input logic types represent other gates such as AND and OR. The logic-based arithmetic processors such as half-adders and half-subtractors are also featured. Naturally, a principal application of the more complex gates is in multi-sensing contexts.
    Topics in current chemistry 01/2011; 300:1-28. DOI:10.1007/128_2010_96 · 4.46 Impact Factor
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    ABSTRACT: The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes.
    Lab on a Chip 05/2010; 10(10):1267-73. DOI:10.1039/b924151e · 6.12 Impact Factor
    ChemInform 02/2010; 30(8). DOI:10.1002/chin.199908031
  • Yumi Makino · Seiichi Uchiyama · Ken-ichi Ohno · Hidetoshi Arakawa
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    ABSTRACT: A novel fluorimetric method for determining radicals using the natural phenol sesamol as a fluorogenic reagent is reported. In this assay, sesamol was reacted with aqueous radicals to yield one isomer of a sesamol dimer exclusively. The dimer emitted purple fluorescence near 400 nm around neutral pH, where it assumed the monoanionic form. This method was applied to the straightforward detection of radical nitric oxide (NO). The ready availability of sesamol should enable rapid implementation of applications utilizing this new assay, particularly in high-throughput analysis or screening.
    Analytical Chemistry 02/2010; 82(4):1213-20. DOI:10.1021/ac9029778 · 5.64 Impact Factor
  • Seiichi Uchiyama · Yumi Makino
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    ABSTRACT: We designed polymeric sensors that created a digital-type fluorescence response to pH variation in an aqueous solution.
    Chemical Communications 06/2009; 19(19):2646-8. DOI:10.1039/b900889f · 6.83 Impact Factor
  • Chie Gota · Kohki Okabe · Takashi Funatsu · Yoshie Harada · Seiichi Uchiyama
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    ABSTRACT: The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.
    Journal of the American Chemical Society 03/2009; 131(8):2766-7. DOI:10.1021/ja807714j · 12.11 Impact Factor
  • Seiichi Uchiyama · Kaoru Iwai
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    Seiichi Uchiyama · Kaoru Iwai · A Prasanna de Silva
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    ABSTRACT: (Figure Presented) Hi-fi mapping: Multiplexing fluorescent sensors that simultaneously target proton concentration and polarity move to micellar nanospaces, self-regulate their positions, and report their pKa values and wavelengths, which are controlled by their local environments. Such sensory functions enable maps of proton gradients near micellar membranes to be drawn.
    Angewandte Chemie International Edition 06/2008; 47(25):4667-9. DOI:10.1002/anie.200801516 · 11.26 Impact Factor
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    ABSTRACT: Fluorescent molecular thermometers showing temperature-dependent fluorescence lifetimes enable thermal mapping of small spaces such as a microchannel and a living cell. We report the temperature-dependent fluorescence lifetimes of poly(NIPAM-co-DBD-AA), which is a random copolymer of N-isopropylacrylamide (NIPAM) and an environment-sensitive fluorescent monomer (DBD-AA) containing a 4-sulfamoyl-7-aminobenzofurazan structure. The average fluorescence lifetime of poly(NIPAM-co-DBD-AA) in aqueous solution increased from 4.22 to 14.1 ns with increasing temperature from 30 to 35 degrees C. This drastic change in fluorescence lifetime (27% increase per 1 degrees C) is the sharpest ever reported. Concentration independency, one of the advantages of fluorescence lifetime measurements, was seen in average fluorescence lifetime (13.7 +/- 0.18 ns) of poly(NIPAM-co-DBD-AA) at 33 degrees C over a wide concentration range (0.005-1 w/v%). With increasing temperature, polyNIPAM units in poly(NIPAM-co-DBD-AA) change their structure from an extended form to a globular form, providing apolar and aprotic environments to the fluorescent DBD-AA units. Consequently, the environment-sensitive DBD-AA units translate the local environmental changes into the extension of the fluorescence lifetime. This role of the DBD-AA units was revealed by a study of solvent effects on fluorescence lifetime of a model environment-sensitive fluorophore.
    The Journal of Physical Chemistry B 04/2008; 112(10):2829-36. DOI:10.1021/jp709810g · 3.30 Impact Factor

Publication Stats

2k Citations
289.46 Total Impact Points


  • 1998–2015
    • The University of Tokyo
      • Department of Pharmaceutical Sciences
      Tōkyō, Japan
  • 2008
    • Queen's University
      • Department of Chemical Engineering
      Kingston, Ontario, Canada
  • 2005–2008
    • Queen's University Belfast
      • School of Chemistry and Chemical Engineering
      Béal Feirste, Northern Ireland, United Kingdom