SIMULTANEOUS MEASUREMENT OF INTRACELLULAR PH AND CA2-SECRETING CELLS BY SPECTRAL IMAGING MICROSCOPY( IN INSULIN)

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 com bined spectral output of furs 2 and SNARF-1 was analyzed to follow cha nges in intracellular Ca2+ concentration ([Ca2+](i)) and intracellular pH (pH(i)) simultaneously and to correct the Ca2+ signal for concurre nt changes in pH(i). Responses of individual hamster insulinoma (HIT-T 15) cells to effecters of ion homeostasis were heterogeneous. Treatmen t with NH4Cl increased pH(i) and transiently increased [Ca2+](i). Remo val of NH4Cl induced cytosolic acidification concomitant with either n o change or sustained increases in [Ca2+](i). Glucose treatment genera lly resulted in rapid and sustained increases in both [Ca2(+)](i) and pH(i) but also heterogeneous pH(i) and [Ca2+](i) responses. Correction s of the fura 2 signal for pH were important for following Ca2+ transi tions elicited by NH4Cl but were less important for glucose-induced re sponses. The spectral imaging microscope provides a sensitive method f or simultaneous measurements of pH(i) and [Ca2+](i) in single cells.