Quantitative imaging of free and total intracellular calcium in cultured cells

Techniques of fluorescence and ion microscopies were combined to study the free [Ca2+] and total Ca in NIH 3T3 fibroblast and L6 rat myoblast cells. Free Ca2+ measurements with the Ca2+ indicator fura-2 and digital imaging reveal an inhomogeneous distribution of free cytoplasmic Ca2+ in both cell lines. Fura-2 also reveals a difference in free Ca2+ activity between the nucleus and cytoplasm of cells. Ion microscopic observations on sister cells show that total Ca in the cytoplasm is also inhomogeneously distributed and that mean cytoplasmic levels of total Ca are higher than levels in the nuclei. In the nuclei of NIH 3T3 cells, the mean free [Ca2+] and total [Ca] were 110 +/- 30 nM and 225 +/- 43 microM, respectively, while regions in the cell cytoplasm contained up to 490 +/- 270 nM free [Ca2+] and 559 +/- 184 microM mean total [Ca]. Intracellular total Ca was greater than 3 orders of magnitude higher than intracellular free Ca2+ in either nuclear or cytoplasmic compartments. Perinuclear cytoplasmic regions in 3T3 cells contained higher free and total Ca than the cell nucleus. Loading of cells with fura-2 did not modify the subcellular distribution of total K, Na, Ca, or Mg. This combination of two powerful ion imaging techniques provides a comparison between free and total calcium in cells and introduces a different approach for examining the role of this important element in cell physiology.