THE DISTRIBUTION OF INTRACELLULAR CALCIUM CHELATOR (FURA-2) IN A POPULATION OF INTACT HUMAN RED-CELLS

Using quantitative fluorescence microscopy of red cells loaded non-dis ruptively with 1-2.5 mmol/l cells of fura-2, we examined the distribut ion of the incorporated free chelator among and within individual cell s. Cytoplasmic hemoglobin quenched the effective fluorescence yield of fura-2 by a factor of about 100. All red cells were found to fluoresc e upon excitation at 380 nm, and the fluorescence intensities they emi tted at 510 nm were approximately +/- 20% about the mean intensity, in dicating a fairly uniform distribution of incorporated chelator among the cells. Red cells loaded with these high levels of fura-2 retained their biconcave shape, and a comparison between their transmission ima ges at 415 nm and their fura-2 fluorescence images suggests that the c oncentration of fura-2 was also uniform throughout the cytosol. These results validate assumptions made in earlier experiments with non-fluo rescent incorporated Ca2+ chelators, and demonstrate the feasibility o f fura-2 and Ca2+ imaging of intact red cells, despite considerable qu enching of probe fluorescence by hemoglobin.