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

Methods Cell Sci. 2003;25(3-4):123-33. doi: 10.1007/s11022-004-2043-8.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aniline Compounds / chemistry
  • Animals
  • Calcium / metabolism*
  • Calibration
  • Cells, Cultured
  • Cytoplasm / metabolism
  • Mice
  • Mice, Transgenic
  • Microscopy, Confocal / methods*
  • Microscopy, Fluorescence / methods
  • Myocardial Contraction / physiology
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / physiology
  • Xanthenes / chemistry


  • Aniline Compounds
  • Xanthenes
  • Fluo-3
  • Calcium