Real time in situ confocal imaging of calcium wave in the perfused whole heart of the rat.

To understand the calcium handling in whole heart having automaticity of the sinus node, we have developed a system of in situ imaging the intracellular calcium ion concentration in the perfused whole heart of the rat. The system consists of a stage-fixed upright microscope equipped with a real-time confocal laser scanning device of a multipinhole type with a water-immersion objective lens for observation. This in situ imaging system rendered observations and analyses of the rapidly changing images of intracellular calcium dynamics possible in the whole rat heart loaded with fluo-3. The scanning was conducted at a video rate of 30 frames per second, and the confocal effects included both X and Y planes. Calcium waves were frequently interrupted by calcium transients from either external electro-stimulation pulses or spontaneous sinus rhythm. Our findings suggest that abnormal calcium waves in minute areas cannot disturb the excitation-contraction coupling in the whole heart if the myocardial cells have orderly end-on-end intercellular electric paths.