Simultaneous measurement of Ca2+, contraction, and potential in 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
H A
HA
Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, Maryland 21224.
Stern
M D
MD
Baartz
G
G
Raffaeli
S
S
Hansford
R G
RG
Talo
A
A
Lakatta
E G
EG
Capogrossi
M C
MC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002469:Cell Separation; D002614:Chelating Agents; D004559:Electric Wiring; D004594:Electrophysiology; D006321:Heart / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials; D008991:Monitoring, Physiologic / Q000295:instrumentation*; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology; D009994:Osmolar Concentration
D002614:Chelating Agents; D007211:Indoles; C059587:Indo-1 pentaacetoxymethyl ester; D002118:Calcium