Application of Real-Time Confocal Microscopy to Observations of ATP-Induced Ca^<2+>-Oscillatory Fluctuations in Intact Corneal Epithelial Cells (Mini-SymposiumCurrent Topics in Cell Biology : From Molecules to Tissues)

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Digital imaging of intracellular calcium ion concentration ([Ca^<2+>]_i) dynamics has been a fundamental technique in cell biology. The present study examines the possibility of using the confocal microscope for [Ca^<2+>]_i imaging of living tissue. We analyzed the ATP-induced [Ca^<2+>]_i dynamics of rabbit corneal epithelium as an experimental model. After loading the cells with Indo-1, a realtime confocal microscope (Nikon RCM/Ab) revealed individual images. ATP (100 μM) in the perfusate elicited an increase in [Ca^<2+>]_i. Superficial cells showed a biphasic change in [Ca^<2+>]_i composed of an initial spike phase followed by a persistent plateau phase. On the other hand, oscillatory fluctuations were evident in the mid-wing cell layer, and they were often synchronized, indicating intercellular communication. The spikes in the wing cells did not affect the [Ca^<2+>]_i dynamics of the superficial cells. In conclusion, the optical slicing effect of confocal microscopy is suitable for observing thick specimens, such as living tissues composed of various cell types. This is the first report to describe [Ca^<2+>]_i dynamics in intact epithelial tissue specimens, and to reveal heterogeneous responses of different cell layers.
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