24] Confocal imaging analysis of intracellular ions in mixed cellular systems or in situ using two types of confocal microscopic systems

Abstract

Publisher Summary The chapter presents a discussion on confocal imaging analysis of intracellular ions in mixed cellular systems or in situ using two types of confocal microscopic systems. To determine changes in [Ca 2+ ] i in mixed cellular systems such as in situ and exact spatial heterogeneity of the Ca 2+ response within single cells, microscopic techniques with a high Z-axis resolution are needed. In this connection, the thin optical sectioning capability of laser scanning confocal microscopy that rejects light from out-of-focus planes, permits imaging of [Ca 2+ ] i in individual cells in situ in optical sections about 1 μ m thick. Confocal imaging analysis permits measurement of [Ca 2+ ] i in individual cells in tissue preparations, such as blood vessels, in situ . Accordingly, it is useful in the characterization of Ca 2+ mobilization in vascular endothelial cells in pathological states such as hypertension and diabetes, which cannot be examined using cultured cells with conventional fluorescence imaging systems. In addition, a high-speed three-dimensional (3-D) confocal imaging system using a multipinhole confocal disk scanner and a high speed cooled CCD camera permits simultaneous imaging of cells in different focal planes at video rates and should be useful for the study of interactions between these cells. Also, confocal imaging technology can apply analysis of other intracellular ions such as Mg 2+ , Na + , K + , H + , and Cl – as well as Ca 2+ using each appropriate ion-sensitive fluorophore. In the near future, improvement of confocal imaging technology with further development of these ion-sensitive fluorophores may permit visualization of activation of an ion channel with high spatiotemporal resolution comparable to those of electrophysiological recording.