Intracellular Ca²⁺ measurements in living cells

Abstract

Intracellular Ca²⁺ acts as the second messenger in a variety of cells. Many cellular functions are tightly regulated by the intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Therefore, measurement of the [Ca²⁺]_i is of critical importance. Calcium-sensitive dual excitations, indicators, such as fura-2, are now widely used to measure the [Ca²⁺]_i in living cells. The development of techniques allowing the measurement of [Ca²⁺]_i has contributed noticeably to our understanding of many cellular functions. Digital video microscopy, confocal laser scanning microscopy, and multiphoton microscopy allow accurate spatial analysis of [Ca²⁺]_i at the subcellular level. Fura-2 has been used commonly to measure the [Ca²⁺]_i because of the sensitivity and specificity of the method. It can be loaded into living cells with little disruption of the cellular functions. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used extracellularly because of its ability to pass through cell membranes, whereas fura-2 by itself cannot be introduced into the cells. For fura-2 measurements, measurements of the fluorescence intensities at two excitation wavelengths can be used to obtain an estimate of the [Ca²⁺]_i, independent of the dye concentration and the thickness of the cell membrane. In this review, I summarize the advantages and pitfalls of using fura-2 and other standard Ca²⁺ indicators, methods used to measure the [Ca²⁺]_i, including simultaneous measurements of cell movements and the changes in the [Ca²⁺]_i, and procedures used for measuring the cellular and/or subcellular Ca²⁺ concentrations in living cells from the cochlea, such as the outer and inner hair cells.