Ratio-Imaging of Calcium Waves in Cultured Hepatocytes Using Rapid Scanning Confocal Microscope and lndo-1

Abstract

We tried to obtain ratio-images of Ca²⁺ responses in cultured rat hepatocytes using indo-1 and newly developed video-rate ultraviolet (UV) laser-scanning confocal microscope systems. Indo-1 was excited by 351 nm laser light, and the emissions at 400—440 nm and over 440 nm were measured. To get precise ratio-images the systems were adjusted to correct chromatic aberrations in the UV range. As a result, the focal points and the depths of field became almost the same at both emission wavelengths. The ratio-images were usually made by averaging eight successive frames to improve the signal-to-noise ratio at 3.75 images/sec. The in situ calibration yielded the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 0.1 μM in nuclear regions and that of 0.2 μM in cytoplasmic regions. Phenylephrine (15 μM) elicited Ca²⁺ oscillations. The increase in [Ca²⁺]_i in each oscillation originated from a specific region near the plasma membrane and propagated to the other side of the cell in 2—4 sec. [Ca²⁺]_i in the nuclear regions also increased to about the same levels as in the cytoplasmic regions. [Ca²⁺]_i then decreased slowly in 10—20 sec. The patterns of Ca²⁺ waves were always the same in the cells studied.