Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium |
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Authors: | RY Tsien TJ Rink |
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Institution: | Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG U.K. |
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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 μm 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 μM 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. |
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Keywords: | Neutral carrier Ion-selective electrode Siliconization tetraphenylborate e m f electromotive force or potential between the ion-sensitive electrode and a reference electrode in the same solution EGTA HEEDTA Mops Hepes Pipes Taps |
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