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

Raul Martínez-Zaguilán, Mark W. Gurulé, Ron Lynch

Research output: Contribution to journalArticle

40 Scopus citations


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 (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi 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 pHi but also heterogeneous pHi 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 pHi and [Ca2+]i in single cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Issue number5 39-5
Publication statusPublished - May 1996



  • Fluorescence
  • Fura 2
  • HIT-T15
  • Microfluorometry
  • SNARF-1

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology
  • Physiology (medical)

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