Development of New Objective Lens with Double Cylinders

新型双筒物镜的研制

• 批准号: 11558087
• 负责人: OHTA Yoshihiro
• 金额: $ 7.62万
• 依托单位: Tokyo University of Agriculture and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558087/
• 关键词: Objective lens; fluorescence microscope; mitochondoria; membrane potential; calcium signaling; multiple staining; CCDカメラ; 2重染色; 細胞; 経時変化; フィルタホイール; 反射ミラー; 暗視野照明

We have developed a florescence microscope by using objective lens with the double cylinders. Since this objective enabled us to detect the time course of fluorescence at different wavelengths simultaneously, we performed the following two experiments, 1) In adrenocortical cells and C6 glioma cells, we observed calcium signaling and mitochondrial membrane potentials. When the intracellular calcium concentration shows a local and rapid increase near mitochondria, the mitochondria show the transient and sudden depolarization, leading to the release of intramitochondrial calcium. When the calcium concentration increases gradually near the mitochondria, however, mitochondria show the uptake of calcium ion and decrease the calcium concentration at cytosol. 2) The changes in mitochondrial membrane potential were detected during the mitosis. The stage of mitosis was identified with Hoechst 33342. The mitochondrial membrane potential decreased during mitosis. On the other hand, the membrane potential recovered after the mitosis. This results might suggest that the intracellular ATP concentration could decrease during mitosis and that it could increase after the mitosis.

我们用双圆柱物镜研制了一台荧光显微镜。由于这一目标使我们能够同时检测不同波长的荧光的时间进程，我们进行了以下两个实验：1)在肾上腺皮质细胞和C6胶质瘤细胞中，我们观察了钙信号和线粒体膜电位。当线粒体附近细胞内钙离子浓度局部快速升高时，线粒体出现短暂和突然的去极化，导致线粒体内钙的释放。然而，当线粒体附近的钙浓度逐渐增加时，线粒体表现出对钙离子的摄取，并降低了胞浆中的钙浓度。2)检测有丝分裂过程中线粒体膜电位的变化。有丝分裂阶段用Hoechst 33342进行鉴定。有丝分裂过程中线粒体膜电位降低。另一方面，有丝分裂后膜电位恢复正常。这一结果可能表明，细胞内的ATP浓度在有丝分裂过程中可能会下降，在有丝分裂后可能会上升。

项目成果

Nakayama et al.: "Fluorescence Imaging of Metabolic Respcnses in Single Mitochondria"Biochem. Biophys. Res. Commun.. 290. 23-28 (2002)

Nakayama 等人：“单线粒体代谢反应的荧光成像”Biochem。

Makoto Yamada: "Dynamic mobility of genetically expressed fusion protein between cytochrome P450IAI and NADPH-cytochrome P450 reductase in yeast microsomes"Biochemistry. 38. 9465-9470 (1999)

Makoto Yamada：“酵母微粒体中细胞色素 P450IAI 和 NADPH-细胞色素 P450 还原酶之间遗传表达的融合蛋白的动态迁移率”生物化学。

Nakayama, et al.: "Fluorescence Imaging of Metabolic Responses in Single Mitochondria"Biochem. Biophys Res. Commun.. 290. 23-28 (2002)

Nakayama 等人：“单线粒体代谢反应的荧光成像”Biochem。

渡辺功一: "蛍光顕微鏡による単離ミトコンドリアの観察方法"生物物理. 224. 258-260 (1999)

Koichi Watanabe：“使用荧光显微镜观察分离线粒体的方法”生物物理学 224. 258-260 (1999)。

Miyuki Hoolahan: "Molecular Steroidogenesis"Universal Academy Press Inc.. 442 (2000)

Miyuki Hoolahan：“分子类固醇生成”Universal Academy Press Inc.. 442 (2000)