A described system enables simultaneous measurement of cytosolic Ca2+ concentration, cell length, and membrane potential or current in single cardiac myocytes without cross talk.
Describes a novel methodology for the simultaneous measurement of calcium transients, contraction, and electrophysiology in isolated cardiac myocytes.
A system is described that can simultaneously record cytosolic Ca2+ concentration (Ca2+i), cell length, and either membrane potential or current in single cardiac myocytes loaded with the fluorescent Ca2+ indicator indo-1. Fluorescence is excited by epi-illumination with 3.8-microsecond flashes of 350 +/- 5 nm light from a xenon arc. Indo-1 fluoresence is measured simultaneously in spectral windows of 391-434 nm and 457-507 nm, and the ratio of indo-1 emission in the two bands is computed as a measure of Ca2+i for each flash. With cells loaded with the permeant acetoxymethyl ester of indo-1, quantitation of Ca2+i is not precise, owing to subcellular compartmentation of indo-1; however, the instrument would allow full quantitation if indo-1 free acid was introduced by microinjection. Simultaneously, cell length is measured on-line from the bright-field image of the cell. Because fluorescence collection is time gated during the brief flash, and red light (650-750 nm) is used for the bright-field image, cell length and Ca2+i measurements are obtained simultaneously without cross talk. Membrane potential or current can be recorded simultaneously with indo-1 fluorescence and cell length via standard patch-clamping techniques.
Spurgeon et al. (Thu,) reported a other. Simultaneous measurement system was evaluated. A described system enables simultaneous measurement of cytosolic Ca2+ concentration, cell length, and membrane potential or current in single cardiac myocytes without cross talk.