Determination of the chelatable iron pool of single intact cells by laser scanning microscopy

We have previously established a method of detecting intracellular chelatab le iron in viable cells based on digital fluorescence microscopy, To quanti fy cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the met hod to confocal laser scanning microscopy, which should allow the determina tion of the indicator concentration on the single-cell level. The fluoresce nt heavy-metal indicator phen green SK (PG SK), the fluorescence of which i s quenched by iron, was loaded into cultured rat hepatocytes, The hepatocel lular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transi tion metal chelator 2,2'-dipyridyl (2,2'-DPD, 5 mM), We optimized the scann ing parameters for quantitatively recording changes in fluorescence and det ermined individual intracellular PG SK concentrations from the unquenched c ellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "c ytosolic" medium. An ex situ calibration method based on laser scanning mic roscopy was set up to determine the concentration of cellular chelatable ir on from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM ). As the stoichiometry of the PG SK:Fe2+ complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from abs olute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 mu M chelatable iron in hepatocytes, This method makes it possible to determine the pool of chelatable iron in single vital cells independently o f cellular differences (e.g., dye loading, cell volume) in heterogeneous ce ll populations. (C) 2000 Academic Press.