Local Application of Active Physiological Substances Using Laser, and Simultaneous Optical Recordings of Intracellular Ca^<++> Concentration and Membrane Potential

使用激光局部应用活性生理物质，同时光学记录细胞内 Ca^< > 浓度和膜电位

基本信息

批准号：
01870006
负责人：
HIRANO Tomoo
金额：
$ 5.38万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B).
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

A fluorescent microscope system which are capable of (1) fast local application of physiologically active substances such as Ca ions or cyclic nucleotide, with the aid of caged molecules and focused laser beam, simultaneous (2) optical measurement of membrane potential and (3) optical measurement of intracellular concentration of Ca ions, have been constructed. (1) Nitrogen laser was used as a source of ultraviolet laser beam, and the light was led into an inverted microscope using optical fiber. The optical fiber was connected to a lens system with a X-Y positioner mounted on a camera tube of the microscope. The light was further focused through a X40 objective lens. (2) Optical recording of membrane potential was performed with potentiometric dies such as RH155 and with a photodiode. Transmitted light from a halogen lamp which passed through filters and a specimen was detected with a photodiode mounted on a lateral tube of the microscope. (3) Fluoresent signals reflecting intracellular Ca concentration was detected by a photomultiplier mounted on a lateral tube of the microscope. Fluo-3 AM was used as an indicator. Epifluoresent light came from Hg arc lamp was reflected off a dicroic mirror and led to a specimen. Electrical signals from a photodiode and a photomultiplier were current-voltage converted, multiplied, A-D converted, stored in a computer and analysed. The constructed system has two problems. One is that only a small portion of UV laser beam reaches a specimen, and most is lost within the light path. Another is the poor spatial resolution. To overcome these problems, I am planning a few changes such as the introduction of laser beam to different site of the microscope, and the increase of number of optical detectors.

荧光显微镜系统，能够（1）借助笼子分子和聚焦激光束，同时（2）膜电位的光学测量和（3）构建了Ca的细胞内浓度的光学测量。（1）将氮激光用作紫外激光束的来源，并使用光纤将光引向倒置显微镜。光纤连接到镜头系统，并在显微镜的摄像头上安装了X-y定位器。光线通过X40物镜进一步聚焦。（2）用电位测量模具（例如RH155和光电二极管）进行膜电位的光学记录。通过安装在显微镜外侧管上的光电二极管检测到通过过滤器和试样的卤素灯传输光。（3）通过安装在显微镜侧管上的光电倍增体检测到反映细胞内Ca浓度的荧光信号。 Fluo-3 AM用作指标。 Espifluoresent Light来自HG ARC灯，反射出二十具镜子，并导致标本。来自光电二极管和光电倍增仪的电信号经过电流电压转换，乘以A-D转换，存储在计算机中并进行分析。构造的系统有两个问题。一个是，只有一小部分紫外线激光束到达标本，并且大部分在光路中丢失。另一个是空间分辨率差。为了克服这些问题，我计划进行一些更改，例如将激光束引入显微镜的不同位点，以及光学探测器数量的增加。