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

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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