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

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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.