1992 Fiscal Year Final Research Report Summary
Development of Fluorescent Microscope Having Multi-Excitation for Simultaneous Measurements of Several Dyes in Order to Investigate Cell Functions
| Project/Area Number |
02557087
|
|---|
| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Physical pharmacy
|
|---|
| Research Institution | Hokkaido 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)
|
|---|
| Project Period (FY) |
1990 – 1992
|
| Keywords | membrane-potential-dependent fluorescent dye / Ultra-violet irradiation / dihydrorhodamine / active oxygen / lipophilic ions / hepatocyte, hepatic cells / allosensitization |
|---|
| 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.
|
