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

James Sham, Christopher Ian Spencer

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationIon Channels in the Pulmonary Vasculature
PublisherCRC Press
Pages733-750
Number of pages18
ISBN (Electronic)9780849350375
ISBN (Print)0824759680, 9780824759681
StatePublished - Jan 1 2005

ASJC Scopus subject areas

  • Medicine(all)

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