A ratiometric two-photon probe for quantitative imaging of mitochondrial pH values

Area of Science:

• Cell Biology
• Biochemistry
• Biomedical Imaging

Background:

• Mitochondrial pH (pHmito) is crucial for cellular function and implicated in various disorders.
• Accurate measurement of pHmito in live systems is essential for understanding these roles.

Purpose of the Study:

• To develop and validate a novel ratiometric two-photon probe (CMP1) for quantitative pHmito imaging.
• To investigate the spatial heterogeneity of pHmito and its dynamic changes in live cells and tissues.

Main Methods:

• Development of a ratiometric two-photon probe (CMP1) based on intramolecular charge transfer from 2-naphthol.
• Quantitative imaging of pHmito in live cells and tissues using two-photon microscopy (TPM).
• Assessment of CMP1's performance, including pKa, emission spectra, loading, selectivity, cytotoxicity, and fluorescence brightness.

Main Results:

• CMP1 has an ideal pKa of 7.86 ± 0.05 and exhibits yellow-to-red emission changes across pH 6.0-9.0.
• The probe allows for selective, robust, and quantitative imaging of pHmito in mitochondria with low cytotoxicity.
• TPM imaging revealed heterogeneous pHmito distribution, with higher values in the perinuclear region.
• Dynamic pHmito changes were observed during CCCP treatment and autophagic processes.
• CMP1 was successfully applied to Parkinson's disease model astrocytes and living hippocampal tissues.

Conclusions:

• CMP1 is a valuable tool for quantitative, in situ imaging of mitochondrial pH in live cells and tissues.
• The probe facilitates the study of pHmito dynamics and spatial heterogeneity in various biological contexts.
• CMP1 holds significant potential for advancing biomedical research, particularly in understanding mitochondrial function and disease.