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Cytometry A. 2003 Apr;52(2):77-89.

Fluorescence lifetime-resolved pH imaging of living cells.

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Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21201, USA.



The regulation and maintenance of intracellular pH are critical to diverse metabolic functions of the living cells. Fluorescence time-resolved techniques and instrumentations have advanced rapidly and enabled the imaging of intracellular pH based on the fluorescence lifetimes.


The frequency-domain fluorescence lifetime imaging microscopy (FLIM) and fluorophores displaying appropriate pH-dependent lifetime sensitivities were used to determine the temporal and spatial pH distributions in the cytosol and vesicular compartment lysosomes.


We found that cytosolic pH levels are different in 3T3 fibroblasts, Chinese hamster ovary (CHO) cells, and MCF-7 cells when using the pH probe carboxy-SNAFL2. We also tracked the transient cytosolic pH changes in the living CHO cells after treatments with proton pump inhibitors, ion exchanger inhibitors, and weak base and acid. The intracellular lysosomal pH was determined with the acidic lifetime probes DM-NERF dextrans, OG-514 carboxylic acid dextrans, and LysoSensor DND-160. Our results showed that the resting lysosomal pH value obtained from the 3T3 fibroblasts was between 4.5 and 4.9. The increase of lysosomal pH induced by the treatments with proton pump inhibitor and ionophores also were observed in our FLIM measurements.


Our lifetime-based pH imaging data suggested that FLIM can measure the intracellular pH of the resting cells and follow the pH fluctuations inside the cells after environmental perturbations. To improve the z-axis resolution to the intracellular lifetime-resolved images, we are investigating the implementation of the pseudo-confocal capability to our current FLIM apparatus.

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