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.

一种荧光显微镜系统，能够 (1) 借助笼状分子和聚焦激光束快速局部应用生理活性物质，例如 Ca 离子或环核苷酸，同时 (2) 膜电位的光学测量，以及 (3)已构建了细胞内 Ca 离子浓度的光学测量方法。 (1)采用氮激光器作为紫外激光束源，利用光纤将光引入倒置显微镜。光纤连接到带有安装在显微镜摄像管上的 X-Y 定位器的透镜系统。光线通过 X40 物镜进一步聚焦。 (2)使用RH155等电位测定芯片和光电二极管进行膜电位的光学记录。来自卤素灯的透射光穿过滤光片和样本，用安装在显微镜侧管上的光电二极管进行检测。 (3)通过安装在显微镜侧管上的光电倍增管检测反映细胞内Ca浓度的荧光信号。 Fluo-3 AM用作指示剂。来自汞弧灯的落射荧光被二色镜反射并引导至样本。来自光电二极管和光电倍增器的电信号经过电流-电压转换、相乘、A-D 转换、存储在计算机中并进行分析。构建的系统有两个问题。一是只有一小部分紫外激光束到达样本，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，我正在计划一些改变，例如将激光束引入显微镜的不同位置，以及增加光学探测器的数量。