Background: 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.Methods: 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.Results: 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.Conclusions: 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. Cytometry Part A 52A:77–89, 2003.

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.