Quantitative profiling pH heterogeneity of acidic endolysosomal compartments using fluorescence lifetime imaging...

Area of Science:

• Cell Biology
• Biophysics
• Physiology

Background:

• The endolysosomal system is vital for cellular homeostasis and organism health.
• Acidic compartment pH is critical for endolysosome function and is influenced by internal and external factors.

Purpose of the Study:

• To present a novel fluorescence lifetime imaging microscopy (FLIM) method for quantitative pH analysis of acidic endolysosomal compartments.
• To demonstrate the method's applicability in diverse mammalian cells and the live organism *Caenorhabditis elegans*.

Main Methods:

• Utilizing fluorescence lifetime imaging microscopy (FLIM) with pH-sensitive dyes for quantitative pH measurements.
• Employing FLIM to analyze pH profiles in endolysosomal compartments across various cell types and in *C. elegans*.
• Measuring rapid pH changes in response to environmental stimuli.

Main Results:

• The FLIM method offers high sensitivity for detecting subtle pH differences and revealing heterogeneity within and between cells.
• The technique allows for rapid pH measurements, avoiding issues with transgenic reporters or variable dye concentrations.
• Absolute pH quantification was achieved, highlighting the importance of pH dynamics.

Conclusions:

• FLIM provides a robust tool for investigating endolysosomal pH dynamics and heterogeneity.
• This method facilitates research into lysosomal functions and their regulation in health and disease.
• The approach is valuable for studying cellular homeostasis and organism fitness.