Publication Date
3-1-2025
Journal
Molecular Biology of the Cell
DOI
10.1091/mbc.E23-06-0220
PMID
39878653
PMCID
PMC11974955
PubMedCentral® Posted Date
2-7-2025
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Lysosomes, Hydrogen-Ion Concentration, Caenorhabditis elegans, Animals, Endosomes, Microscopy, Fluorescence, Humans, HeLa Cells, Mice
Abstract
The endolysosomal system plays a crucial role in maintaining cellular homeostasis and promoting organism fitness. The pH of its acidic compartments is a crucial parameter for proper function, and it is dynamically influenced by both intracellular and environmental factors. Here, we present a method based on fluorescence lifetime imaging microscopy (FLIM) for quantitatively analyzing the pH profiles of acidic endolysosomal compartments in diverse types of primary mammalian cells and in live organism Caenorhabditis elegans. This FLIM-based method exhibits high sensitivity in resolving subtle pH differences, thereby revealing heterogeneity within a cell and across cell types. This method enables rapid measurement of pH changes in the acidic endolysosomal system in response to various environmental stimuli. Furthermore, the fast FLIM measurement of pH-sensitive dyes circumvents the need for transgenic reporters and mitigates potential confounding factors associated with varying dye concentrations or excitation light intensity. This FLIM approach offers absolute pH quantification and highlights the significance of pH heterogeneity and dynamics, offering a valuable tool for investigating lysosomal functions and their regulation in various physiological and pathological contexts.