Development of Fluorescence Microscopy for Visualizing Single Biomolecules in Living Cells

用于可视化活细胞中单个生物分子的荧光显微镜的发展

• 批准号: 10558110
• 负责人: FUNATSU Takashi
• 金额: $ 8.32万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10558110/
• 关键词: Single molecule imaging; Fluorescence microscopy; mRNA; Chaperonin

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were 〜10 sec for TMR and 〜2 sec for IC5 under the incident laser power of 0.5 W/mm^2. Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm^2. 2D sliding movement of single kinesin molecules along microtubules on glass surface, and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.

单荧光分子在水溶液中成像的第一次在视频速率使用Nipkow盘型共聚焦显微镜。该方法的性能进行了评估，通过成像单一驱动蛋白分子标记的荧光染料四甲基罗丹明（TMR）或IC 5。在0.5 W/mm^2的入射激光功率下，TMR和IC 5的荧光团的光分解寿命分别为10秒和12秒。在0.65 ~ 3 W/mm^2的激光功率范围内，荧光强度和光漂白速率均与激光功率成正比。利用该显微镜可以观察到单个驱动蛋白分子在玻璃表面沿着微管的二维滑动运动以及单个驱动蛋白分子在粘性溶液中的三维布朗运动。这些结果表明，该方法可用于活细胞单分子事件的真实的实时研究。

项目成果

Ishii, Y. et al.: "Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous"Chemical Physics. 247. 163-173 (1999)

Ishii, Y. 等人：“附着在水性卷曲螺旋蛋白上的单个荧光团之间的荧光共振能量转移”化学物理学。

船津高志: "1分子の生体高分子を観て操る"高分子. 48. 906-909 (1999)

Takashi Funatsu：“观察和操纵单分子生物聚合物”聚合物 48. 906-909 (1999)

Y.Ishii et al.: "Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous solution"Chemical Physics. 247. 163-173 (1999)

Y.Ishii 等人：“水溶液中卷曲螺旋蛋白上附着的单个荧光团之间的荧光共振能量转移”化学物理学。

Tashiro,K. et al.: "Design and simulation of particles and biomolecules handling micro flow cells with three-dimensional sheath"Micro Total Analysis Systems 2000. 209-212 (2000)

田代，K.

田口英樹 ら: "シャペロニンとGFPフォールディング"生物工学会誌. 78. 384-386 (2000)

Hideki Taguchi 等：“Chaperonin 和 GFP 折叠”日本生物工程学会杂志 78. 384-386 (2000)。