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 fura 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 effectors 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.