Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy

Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of furs 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+](i)) and intracellular pH (pH(i)) simultaneously and to correct the Ca2+ signal for concurrent changes in pH(i). Responses of individual hamster insulinoma (HIT-T15) cells to effecters of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pH(i) and transiently increased [Ca2+](i). Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2+](i). Glucose treatment generally resulted in rapid and sustained increases in both [Ca2(+)](i) and pH(i) but also heterogeneous pH(i) and [Ca2+](i) responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pH(i) and [Ca2+](i) in single cells.