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Development of Fluorescent Microscope Having Multi-Excitation for Simultaneous Measurements of Several Dyes in Order to Investigate Cell Functions

Research Project

Project/Area Number 02557087
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Physical pharmacy
Research InstitutionHokkaido University

Principal Investigator

KAMO Naoki  Faculty of Pharmaceutical Sciences, Hokkaido University, Professor, 薬学部, 教授 (10001976)

Co-Investigator(Kenkyū-buntansha) KAIFU Katsumasa  Basic Research Inst.of Oki Electronics, Section Chief, 基盤技術研究所, 研究主任
RIKUKAWA Koji  Dept.Opt-electronics, Nikon Co., Vice Dept.Chief, 光機設計部, 次長
KOIZUMI Yohko  Medical Hospital, Hokkaido University, Lecturer, 医学部・付属病院, 講師 (60113552)
MATSUMOTO Kenji  Faculty of Pharmaceutical Sciences, Hokkaido University, Research Associate, 薬学部, 助手 (80183953)
上田 哲男  北海道大学, 薬学部, 助教授 (20113524)
海部 勝晶  沖電気基盤技術研究所, 研究主任
Project Period (FY) 1990 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥10,700,000 (Direct Cost: ¥10,700,000)
Fiscal Year 1992: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1990: ¥5,000,000 (Direct Cost: ¥5,000,000)
Keywordsmembrane-potential-dependent fluorescent dye / Ultra-violet irradiation / dihydrorhodamine / active oxygen / lipophilic ions / hepatocyte, hepatic cells / allosensitization / 同種棚原感作 / 蛍光色素、 / デヒドロロ-ダミン、 / ロ-ダミン、 / 活性酸素、 / 遊離肝細胞、 / 膜電位、 / 脂溶性イオン、
Research Abstract

We made a microscope where two excitation wavelengths were changed alternatively. The image was digitized and stored in a micro-computer. With combination of this instrument and various fluorescent dyes that can report various quantities such as membrane potential and ionic concentrations, the simultaneous measurement of these quantities becomes feasible and may help the understanding of the mechanism of cell functions.
(1) The method to detect intracellular H_2O_2 formation using dihydrorhodamine 123 was developed. With this method together with the microscope, we determined ultra-violet action spectrum for H_2O_2 formation in epidermal keratinocytes.
(2) One of current problems in transfusion is to prevent allosensitization by UV irradiation. The mechanism, however, is not clear. We measured active oxygen formation by UV irradiation in various blood cells, as well as change in intracellular Ca concentration and membrane potential with the microscope.
(3) One cannot measure the membrane potential of eucaryotic cells with a positively changed probe, because of high accumulation into mitochondria. We devised the correction method and applied to measure the membrane potential of hepatic cells.
(4) With a negatively charged fluorescent dye (merocyanine 540), we failed to measure the membrane potential of hepatic cells, suggesting the necessity of further study on the interaction of a dye and cell membrane.

Report

(4 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] M.MURATSUGU: "Plasma-polymerized allylamine film used as a new solid phase in immunoradiometrric assay(IRMA) I.Modification of glass surface." J.Colloid Inter.Sci.147. 378-386 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] S.KUROSAWA: "Adsorption of anti-human IgG to plasma-polymerized allylamine film formed on a silver plate." Polymers for advanced technologies. 2. 253-259 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M.MURATSUGU: "Plasma-polymerized allylamine film used as a new solid phase in immunoradiometric assay (IRMA) II.Comparison with a polyvinyl chloride plate and effect of antibody (F(ab')^2fragment) concentration on dose-response." Chem.Pharm.Bull.40. 501-503 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] S.SAITHO: "Measurement of plasma membrane potenial in isolated rat hepatocytes using the lipophilic cation,tetraphenyl-phosphonium." Biochim.Biophys.Acta. 1111. 221-230 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] H.SAKURADA: "Use of dihydrorhodamine 123 for detecting intracellular generation of peroxides upon UV irradiation in epidermal keratinocytes." Arch.Dermatol.Res.284. 114-116 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Makoto Muratsugu, Shigeru Kurosawa and Naoki Kamo.: "Plasma-polymerized allylamine film used as a new solid phase in immunoradiometric assay (IRMA) I. Modification of glass surface." J.Colloid Inter. Sci.147. 378-386 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Shigeru Kurosawa, Makoto Muratsugu and Naoki Kamo.: "Adsorption of anti-human IgG to plasma-polymerized allylamine film formed on a silver plate." Polymers for advanced technologies. 2. 253-259 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Makoto Muratsugu, Shigeru Kurosawa, Yoshihito Mori and Naoki Kamo.: "Plasma-polymerized allylamine film used as a new solid phase in immunoradiometric assay (IRMA) II. Comparison with a polyvinyl chloride plate and effect of antibody (F(ab^1)_2 fragment) concentration on dose-response in two-site IRMA." Chem. Pharm. Bull.40. 501-503 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Shyuichi Saitho, Seiji Miyauchi, Yusuke Murakami and Naoki Kamo.: "Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation, tetraphenylphosphonium : correction of probe intracellular binding and mitochondrial accumulation." Biochim. Biophys.Acta 1111. 221-230 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] H.Sakurada, H.Koizumi, A.Ohkawara, T.Ueda and N.Kamo.: "Use of dihydrorhodamine 123 for detecting intracellular generation of peroxides upon UV irradiation in epidermal keratinocytes." Arch. Dermatol. Res.284. 114-116 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M. Muratsugu: "Plasma-polymerized allyamine film used as a new solid phase in immunoradiometric assay (IRMA) 11. Comparison with a polyvinyl chloride plate and effect of antibody(F(ab)_2fragmet)concentration on dose-response." Chem. Phharm.Bull.40. 501-503 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] S. Saitho: "Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation,tetraphenyl-phosphonium." Biochim. Biophys Acta. 1111. 221-230 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] H. Sakurada: "Use of dihydrorhodamine 123 for detecting intracellilar generateon of peroxeides upon UV irradiation in epidermal keratinocytes." Arch. Dermatol. Res.284. 114-116 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] T.Takahashi: "Detection of peroxide in cells by UV-irradiation with dehydrorhodamin" Archives of Biochemistry and Biophysics.

    • Related Report
      1991 Annual Research Report
  • [Publications] 一木 浩: "紫外線照射の血球細胞への効果" 日本輸血学会誌.

    • Related Report
      1991 Annual Research Report
  • [Publications] S.Miyauchi: "In archaebacteria,there is a doxorubicin efflux pum similar to mammalian p-glycoprotein" Biochimica et Biophysica Acta.

    • Related Report
      1991 Annual Research Report
  • [Publications] Y.Imamoto: "Photoreaction cycle of phoborhodopsin studied by low-temperature spectrophotometry" Biochemistry. 30. 7416-7424 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] S.Kurosawa: "Characteristics of sorption of various gases to plasma-polymerized copper phthalocyanine" Langmuir. 8. 254-256 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] E.Tawara: "glucose transport of Haloferax volcanii requires the Na-electrochemical potential gradient" Biochimica et Biophysica Acta. 1070. 293-299 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 須貝 新太郎: "生物物理工学" アイピ-シ-, 251 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] J.Takano et al.: "Action spectrum of peroxidation production in keratinocyte" Photochem.Photobiol.

    • Related Report
      1990 Annual Research Report
  • [Publications] J.Sakurada et al.: "New methods for detection of peroxidation production in living cells by ultraーviolet radiation" Archiv.Biochem.Biophys.

    • Related Report
      1990 Annual Research Report
  • [Publications] S.Miyauchi et al.: "Determination of membrane potential of hypatocycte with use of lipophilic cation" Archiv.Biochem.Biophys.

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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