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Real-time in situ two-photon imaging of intercellular signaling in sinoatrial node

窦房结细胞间信号传导的实时原位双光子成像

基本信息

批准号：
09557022
负责人：
TAKAMATSU Tetsuro
金额：
$ 7.23万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557022/
关键词：
Sinoatrial node Fluo-3 Laser scanning microscopy Calcium ion Digital image Two-photon excitation Multi-pinhole scanner 二光子 in situ イメージング

项目摘要

The confocal microscopy has become a dependable tool for obtaining not only topological information, but also densitometric information. However, there remain many problems with the confocal microscope such as slow scanning speed, mismatched refractive indices, cell injury by the laser beam, bleaching and the penetrability of the fluorescent probe. To improve these problems, this project investigated in situ real-time imaging of intracellular calcium ion concentration ([Ca^<2+>]_i) in the cardiac ventricle or sinoatrial node by using two-photon microscopy.We have developed a new laser scanning microscope system equipped with a microlens-arrayed multi-pinhole scanning disc, water-immersion objective lenses and a mode-locked Ti : sapphire pulse laser, which provide a scanning beam. The new system allows in situ quantitative imaging of non- homogenous changes of the three-dimensional [[Ca^<2+>]_i in a living whole heart, such as calcium wave at a full video rate. Calcium waves propagating from cell to cell were frequently interrupted by calcium transients from spontaneous sinus rhythm. But serial images obtained have low signal-to-noise and cell injury with laser light still remained unsolved.The evolution of the confocal microscope with high spatial and temporal resolution renders the investigation of biological phenomena even in the living organ.

共焦显微镜已成为一种可靠的工具，不仅获得拓扑信息，而且密度信息。然而，共聚焦显微镜仍然存在许多问题，如扫描速度慢，折射率不匹配，激光束损伤细胞，漂白和荧光探针的穿透性。为了改善这些问题，本项目研究了细胞内钙离子浓度的原位实时成像利用双光子显微镜测量了心室或窦房结内[Ca^<2+>]_i的变化.我们研制了一种新的激光扫描显微镜系统，该系统配备了微透镜阵列多针孔扫描盘、水浸物镜和锁模Ti：蓝宝石脉冲激光器，其提供扫描光束。新系统允许在活体全心脏中的三维[[Ca^2+] i的非均匀变化的原位定量成像，例如以全视频速率的钙波。从细胞到细胞的钙波传播经常被自发性窦性心律的钙瞬变中断。但是，激光对细胞的损伤和连续图像的信噪比低等问题一直没有得到很好的解决，随着共聚焦显微镜的发展，高空间和时间分辨率的共聚焦显微镜使生物现象的研究甚至可以在活体器官中进行。