[High-resolution imaging of intracellular calcium ion concentration using rapid scanning confocal microscopy]

Recently, the technique for high time- and spatial-resolution imaging of intracellular calcium ion concentration ([Ca2+]i) has been developed using rapid scanning confocal microscopy to investigate calcium dynamics in restricted areas of cells. Here, the techniques are summarized and the images obtained are introduced. For such imaging, the development of fast scanning confocal microscopes was indispensable. Therefore, the UV-applicable video-rate (30 frames/sec) scanning confocal microscope in which a resonant glavanometer scanner is used for fast horizontal scans has been developed. The microscope enabled us to obtain ratio-images of [Ca2+]i using indo-1, a ratio-imaging probe. The key to success in high time- and temporal-resolution imaging is to find the best probe-loading condition, which depends on the probes and the cells. It is also very important to increase the signal-to-noise ratio value without any effects on the cells. Using the technique, we have succeeded in kinetic analysis of calcium waves in cultured hepatocytes. We have also succeeded in determining the relationship between calcium sparks to calcium transients in excitation-contraction coupling.