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

Am J Physiol. 1996 May;270(5 Pt 1):C1438-46. doi: 10.1152/ajpcell.1996.270.5.C1438.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Benzopyrans
  • Calcium / metabolism*
  • Cricetinae
  • Fluorescent Dyes
  • Fluorometry
  • Fura-2
  • Glucose / pharmacology
  • Hydrogen / metabolism*
  • Hydrogen-Ion Concentration
  • Insulin / metabolism*
  • Insulin Secretion
  • Intracellular Membranes / metabolism*
  • Naphthols
  • Rhodamines
  • Time Factors
  • Tumor Cells, Cultured


  • Benzopyrans
  • Fluorescent Dyes
  • Insulin
  • Naphthols
  • Rhodamines
  • seminaphthorhodaminefluoride
  • Hydrogen
  • Glucose
  • Calcium
  • Fura-2