Methods in Cell Science

, Volume 25, Issue 3–4, pp 123–133 | Cite as

Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology



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 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 interpretaton and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be used to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.


Calcium Calibration Cardiomyocytes Confocal microscopy 





ethylenebis (oxyethylenenitrilo) tetraacetic acid;




photomultiplier tube;


tropomodulin overespressing transgenic


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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  1. 1.SDSU Heart Institute, Department of BiologySan Diego State UniversitySan DiegoUSA
  2. 2.SDSU heart Institute, Department of BiologySan Diego State UniversitySan DiegoUSA

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