Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium

Abstract

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca2+] levels. Pipettes of tip outer diameter down to 0.4 micron were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204-206) but with very hydrophobic cations replacing Na+ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca2+] levels. Lowering the Ca2+ concentration in the internal electrolyte also increased electrode lifetime. Electrodes showed a Nernstian response to [Ca2+] down to 1 micro M free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca2+. Selectivity for Ca2+ over Mg2+ and H+ was sufficiently high that typical free intracellular levels of Mg2+ and H+ caused negligible interference. Selectivity for Ca2+ over Na+ was adequate for cells with 10(-2) M free Na+, but higher levels could raise significantly the detection limit for Ca2+. Preliminary measurements of [free Ca2+] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca2+ microelectrodes and optical indicators are discussed.