SIMULTANEOUS IMAGING OF INTRACELLULAR [CA2-CELLS(] AND PH IN SINGLE MDCK AND GLOMERULAR EPITHELIAL)

The interrelationships between changes in intracellular calcium concen tration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney ce lls and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence vide o microscope. Cells were double labeled with indo 1 and SNARF 1 dyes a nd were excited simultaneously at 350 and 540 nm. Images at four emiss ion wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH bet ween 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were u sed to correct the pH-dependent changes in the calculation of local ce ll calcium. NH4Cl loading produced the expected alkalinization and a c oncurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells be came acidic, a second rise in [Ca2+]i was recorded. Both changes in [C a2+], were from intracellular stores since they persisted in the absen ce of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.