Study for the dynamics of the intracellular Ca concentration changes with high spatial and temporal resolution.

研究具有高空间和时间分辨率的细胞内 Ca 浓度变化的动态。

基本信息

批准号：
02557007
负责人：
OHMORI Harunori
金额：
$ 6.14万
依托单位：
National Insitute for Physiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

We have made an instrument for exchanging two excitation wavelengths for fluorescence dye at the vide-rate by utilizing a galvano-mirror device, combining with a CCD TV camera and a frame processor. By this device, we could record fura-2 fluorescence changes at two excitation wavelengths at 33 msec time intervals to monitor intracellular Ca ionic concentration changes. These paired fluorescence image data give the profile of intracellular Ca ionic concentration changes at the rate of 66 msec. The device is made from two part. The first part is the galvano-mirror device itself and switches the two excitation wavelengths. The second part is a control logic circuit for synchronizing the galvano-mirror device with the image recording CCD camera, and the frame processing system. Since a single frame of a video-image is made of two interlacing fields, the CCD camera is shutter opened for only 15msec during the two component fields are superimposed. The reduction of the light intensity on the … More camera-head is compensated by using an image intensifier coupled CCD camera. It was simple to synchronize all three component devices (the galvano-mirror device, the CCD camera, and the frame processor), but the attempt for image integration for improving the signal to noise ratio of the fluorescence images was relatively hard to be achieved. We have overcome the problem by adopting an integrated circuit for dividing a clock rate and by synchronizing the start of the frame integration with the start of galvano-mirror device.We have not yet applied this device extensively for the physiology experiments. However, we have already succeeded in recording intracellular Ca ionic concentration changes from cultured neurons from the brain stem of a chick, in both at the ordinary TV rate imaging mode and at a slower rate mode with the frame integration. This chopper device is basically vibration-free, and it is expected to be applied in the electrophysiology experiments to record intracellular ionic concentration changes simultaneously with the electrical activity of the cell. Less

我们制作了一种仪器，通过利用电镜装置，结合CCD电视摄像机和帧处理器，以视频速率交换荧光染料的两个激发波长，通过该装置，我们可以记录fura-2荧光变化。两个激发波长以 33 毫秒的时间间隔监测细胞内 Ca 离子浓度的变化。这些配对的荧光图像数据给出了细胞内 Ca 离子浓度以 66 毫秒的速率变化的情况。该装置由两部分组成。第一部分是电镜装置本身并切换两个激发波长，第二部分是用于使电镜装置与图像记录CCD相机以及帧处理系统同步的控制逻辑电路。视频图像由两个交错场组成，在两个分量场叠加期间，CCD 摄像机的快门打开时间仅为 15 毫秒。通过使用以下方法来补偿摄像机头上的光强度减少。图像增强器耦合 CCD 相机很容易同步所有三个组件设备（电镜设备、CCD 相机和帧处理器），但尝试进行图像集成以提高图像荧光的信噪比。这是相对难以实现的，我们通过采用集成电路来划分时钟频率并将帧集成的启动与电镜设备的启动同步来克服了这个问题。我们还没有将该设备主要用于生理然而，我们已经成功地记录了小鸡脑干培养神经元的细胞内钙离子浓度变化，无论是在普通电视速率成像模式还是在帧集成的较慢速率模式下。无振动，基本上有望应用于电生理学实验中，记录细胞内离子浓度的变化与细胞的电活动同步。