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

本文报道了一种荧光显微镜系统，它能（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如钙离子或环核苷酸，（2）同时光学测量膜电位和（3）光学测量胞内钙离子浓度。(1)使用氮激光器作为紫外激光束的光源，并使用光纤将光引入倒置显微镜。将光纤连接到具有安装在显微镜的摄像管上的X-Y定位器的透镜系统。光通过X40物镜透镜进一步聚焦。(2)膜电位的光学记录是用电位芯片如RH 155和光电二极管进行的。从卤素灯透射的光，通过过滤器和一个样本被检测与安装在显微镜的侧管的光电二极管。(3)通过安装在显微镜的侧管上的光电倍增管检测反映细胞内Ca浓度的连续信号。Fluo-3 AM用作指示剂。从汞弧灯发出的出射荧光经双色镜反射后照射到样品上。光电二极管和光电倍增管发出的电信号经电流-电压转换、倍增、模数转换后存入计算机并进行分析。所构建的系统存在两个问题。一是只有一小部分紫外激光束到达样品，大部分在光路中损失;二是空间分辨率差。为了克服这些问题，我计划进行一些改变，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。

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.