Practical aspects of confocal imaging of Ca²⁺ sparks and cytoplasmic Ca²⁺ concentration

In the past two decades, the understanding of intracellular Ca2+ homeostasis has advanced at an unprecedented pace, owing to the revolutionary discovery of Ca2+-sensitive fluorescence indicator dyes and the tremendous progress in cell imaging techniques. Researchers are now able to study subcelullar Ca2+ regulation by resolving minute Ca2+ signals such as Ca2+ sparklets at the submicrometer level (1) as well as imaging [Ca2+] within intracellular organelles such as mitochondria and sarcoplasmic=endoplasmic reticulum (2-4). These technological breakthroughs allow us to begin to appreciate how the local [Ca2+] conveys target-specific messages in an amplitude-and frequency-dependent manner (5) and how these local processes blend into the mosaic of cytosolic Ca2+ regulation and gene expression (69). The local Ca2+ release event or “Ca2+ spark” that originates from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is one of the most thoroughly studied local Ca2+ events. It is the elementary event underlying global Ca2+ transients in striated muscles and an important modulator of membrane potential in smooth muscles. Ca2+ sparks have been recently identified and characterized in pulmonary arterial smooth muscle cells (10-14). However, most of their physiological functions in the regulation of pulmonary vascular reactivity are yet to be determined. In this chapter, we provide an introduction to Ca2+ spark measurement by discussing (1) the general aspects of Ca2+ fluorescent indicator dyes and their calibration, (2) the detection of Ca2+ sparks using laser scanning confocal microscopy, and (3) the basic analysis of Ca2+ spark properties. We hope this will lead to wider application of the technique in the field of pulmonary circulation.