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Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

Published online by Cambridge University Press:  12 May 2005

David M. Plank  and
Mark A. Sussman
David M. Plank
Affiliation:
SDSU Heart Institute, Department of Biology, San Diego State University, San Diego, CA 92182, USA
Mark A. Sussman
Affiliation:
SDSU Heart Institute, Department of Biology, San Diego State University, San Diego, CA 92182, USA
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Abstract

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Keywords

confocal microscopycalciumcardiomyocytescalibration
Type
Research Article
Information
Microscopy and Microanalysis , Volume 11 , Issue 3 , June 2005 , pp. 235 - 243
Copyright
© 2005 Microscopy Society of America

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