Quantitative single-cell fluorescence imaging of indicator dyes

Abstract

The use of fluorescent molecular probes in combination with quantitative optical microscopy has burgeoned in the past few years due to dramatic advances in both fluorophore design and imaging instrumentation. A number of probes that exhibit fluorescence spectral shifts with ligand binding have been synthesized. Among them is fura-2, which has a high binding affinity for calcium ion and which shows an excitation spectral shift between the calcium-bound and calcium-unbound states. This talk will focus on fura-2-loaded arterial smooth muscle cells (SMC) stimulated to produce temporally and spatially dynamic changes in free cytosolic calcium ion concentration ([Ca2+]i) as measured by a charge-coupled device imaging system.Intracellular Ca2+ is known to play a critical role in the regulation of the contractile state of vascular SMCs. A variety of extracellular agonists have been shown to stimulate transient [Ca2+]i elevation in vascular SMCs; among them are nucleotides that activate specific cell surface receptors.