Development of Ultra-high Power Video Microscope for Patch Clamp Study.

用于膜片钳研究的超高功率视频显微镜的开发。

• 批准号: 01880028
• 负责人: SOKABE Masahiro
• 金额: $ 6.21万
• 依托单位: Nagoya 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01880028/
• 关键词: Patch clamp; Video microscopy; Simultaneous measurement; Manipulator; Image analysis; 超高倍率光学顕微鏡; マルチ計測; 膜容量; ビデオマイクロスコピ-; 超高倍率顕微鏡; 膜容量測定

The aim of this research project is to develop a system by which we can make simultaneous measurements of patch shape, single channel currents, patch capacitance, and pipette pressure. The system is composed of ultra-high power differential interference video microscope, high speed oil driven pressure generator, drift free mechanical manipulator, patch clamp amplifier, phaselock amplifier, and 386 based personal computer with frame grabber and A/D, D/A converter. The specification of each component is as follows.1. Video microscope : A modified inverted light microscope (Olympus IMT-II) with video gave a total magnification of 15nm/pixel.2. Pressure generator : Oil driven pressure generator with servo controller gave a rise time of 2 msec and a dynamic range of <plus-minus> 200mmH3. Micromanipulator : Drift-free (0.2 mum/5 min) micro manipulator was constructed from motorized sliders combined with pulse-width and height modulated servo controller.4. Current and capacitance measurement : 1 Sinusoidal wave was fed into the command input of the patch clamp amplifier and the resulting output was separated into single channel currents (0.1 pA) and capacitive currents (0.1 fF), the latter of which was fed into the phaselock amplifier.5. Image processing : 386 based personal computer with a frame grabber (DT2851) was used to acquire patch images. A SIMPLE TIME CODE decorder was constructed to grab images of arbiterary frames.6. Data acquisition : A/D. D/A converter was used to aquire multi-channel electrical signals. The time code was used to synchronize images with electrical signals.By using this system we could visualize patch membranes and get tension vs. activity relationship of stretch activated channels.

本研究的目的是开发一个系统，通过该系统，我们可以同时测量贴片形状，单通道电流，贴片电容和移液管压力。该系统由超高功率差动干涉视频显微镜、高速油压发生器、无漂移机械手、膜片钳放大器、锁相放大器、带图像采集卡和A/D、D/A转换器的386单片机等组成。各部件的规格如下：1.视频显微镜：带视频的改良倒置光学显微镜（Olympus IMT-II）的总放大倍数为15 nm/像素。压力发生器：带伺服控制器的油驱动压力发生器的上升时间为2 msec，动态范围为<plus-minus>200 mmH 3。显微操作器：无漂移（0.2 μ m/5分钟）微操作器是由电动滑块与脉宽和高度调制的伺服驱动器相结合.电流和电容测量：1将正弦波馈入膜片钳放大器的命令输入，并将所得输出分离成单通道电流（0.1 pA）和电容电流（0.1 fF），后者馈入锁相放大器。图像处理：使用基于386的具有帧采集卡的个人计算机（DT 2851）来获取斑块图像。构造了一个SIMPLE TIME CODE解码器，用于抓取任意帧的图像.数据采集：A/D。采用D/A转换器采集多路电信号。利用时间编码技术实现图像与电信号的同步，实现膜片膜的可视化，获得膜片膜牵张激活通道的张力-活性关系。

项目成果

曽我部正博: "機械受容器変換・符号化機序におけるcAMPの関与" 生物物理. 29. 111-117 (1989)

Masahiro Sogabe：“cAMP 参与机械感受器转导和编码机制”生物物理学 29. 111-117 (1989)。

Sokabe,M.: "Electrophysiological analysis of structural aspects of voltageーdependent SR K channel." Comp.Biochem.Physiol.

Sokabe, M.：“电压依赖性 SR K 通道结构方面的电生理学分析。”Comp.Biochem.Physiol。

Sokabe,M.: "Effects of cyclic nucleotides and calcium on transduction and encoding processes in frog muscle spindle" Brain Res.443. 254-260 (1988)

Sokabe,M.：“环核苷酸和钙对青蛙肌梭转导和编码过程的影响”Brain Res.443。

Sachs,F.: "Stretch activated ion channels and membrane mechanics." Neurosci.Res.12. S1-4 (1990)

Sachs,F.：“拉伸激活离子通道和膜力学。”

Sokabe, M.: "Towards molecular mechanism of the gating in stretch activated channels." Comparative Aspect of Mechano-Reception.

Sokabe, M.：“探讨拉伸激活通道中门控的分子机制。”