|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1990 : ¥1,000,000 (Direct Cost : ¥1,000,000)
I have developed a dual, digital fluorescence imaging system and new confocal laser scanning microscope employing an inverted optical system for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca^<2+>]_i) in heart muscle cells.
In 1990, Images of[Ca^<2+>]_i taken at 67-ms intervals during propagation of calcium waves revealed that the calcium wave front was constant in shape, spatially steep, typically rising from 500 to 1200 nM in about 10 mu m, and propagating at constant velocity, typically 100 mu m/sec at 22 ﾟC. In 1991, I have investigate the biological application of a confocal laser scanning microscope for imaging the calcium wave. This instrument has several advantages for observing moving objects and flexible choice of scanning modes. 1) A faster scanning speed of up to 0.25 sec per frame including line scan, fast horizontal and vertical full-frame scan and a faster horizontal narrow band scan. 2) An efficient optical path specially designed for th
e inverted fluorescence microscope system. 3) Direct transfer of digitized images from the frame memory in the CLSM to that of an image processor, avoiding the degradation of densitometric data by duplicated A/D and D/A conversion. These features gave us the opportunity to investigate the properties of the calcium wave during its propagation between isolated guinea-pig ventricular cell pairs. The actual[Ca^<2+>]_i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of[Ca^<2+>]_i in optically sectioned unit volumes of the cell. Optical sectioning images obtained enable us to determine the cell-to-cell junction between heart muscle paired cells and reveal that the high[Ca^<2+>]_i can evoke calcium-induced calcium release from the sarcoplasmic reticulum in the neighboring heart muscle cells through the gap junction without delay.
The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living heart muscle cells such as the calcium wave, a model for arrhythmia at cellular level.