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Updated: Dec 24, 2025
Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Published on: January 24, 2017
A colorimetric and ratiometric fluorescent probe for ClO- targeting in mitochondria and its application in vivo
Hongde Xiao1, Jianhui Li, Jin Zhao
1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. yingui@nju.edu.cn.
Abstract:
A colorimetric and ratiometric fluorescent probe PMN-TPP for imaging mitochondrial ClO- was prepared. The selectivity of PMN-TPP was excellent and the detection would not be influenced by other ROS. The limit of detection (LOD = 3σ/slope) for ClO- was evaluated to be 0.43 μM, suggesting the probe's high sensitivity to ClO-. For the biological applications, PMN-TPP performed well in detecting endogenous HClO in living RAW264.7 macrophage cells. A co-localization study employing Mito Tracker green revealed that PMN-TPP was specifically located in the mitochondria of living RAW264.7 macrophage cells. In the in vivo experiment, a nude mouse with acute inflammation stimulated by lipopolysaccharide (LPS) was employed. After injection of PMN-TPP, the fluorescence signal changed gradually in 30 min and then remained unchanged in the injection region, demonstrating that PMN-TPP could detect the endogenous HClO in living animals.
Insights
A novel fluorescent probe, PMN-TPP, was developed for sensitive detection of hypochlorite (ClO-) in mitochondria. This probe successfully imaged ClO- in living cells and animals, demonstrating its potential for biological applications.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Cell Biology
Background:
- Hypochlorite (ClO-) is a reactive oxygen species involved in cellular signaling and inflammation.
- Accurate detection of ClO- in biological systems is crucial for understanding its role in disease.
- Existing probes may lack specificity or sensitivity for mitochondrial ClO- imaging.
Purpose of the Study:
- To develop a selective and sensitive fluorescent probe for imaging mitochondrial hypochlorite (ClO-).
- To evaluate the probe's performance in biological samples, including living cells and animals.
- To investigate the probe's localization and application in inflammatory models.
Main Methods:
- Synthesis and characterization of the PMN-TPP probe.
- Spectroscopic analysis to determine probe selectivity and sensitivity for ClO-.
- Confocal microscopy for co-localization studies with Mito Tracker Green in RAW264.7 cells.
- In vivo fluorescence imaging in lipopolysaccharide (LPS)-induced inflamed nude mice.
Main Results:
- PMN-TPP exhibited excellent selectivity for ClO- over other reactive oxygen species.
- The probe demonstrated high sensitivity with a limit of detection of 0.43 μM.
- PMN-TPP was successfully localized to mitochondria in living RAW264.7 cells.
- In vivo imaging showed dynamic changes in fluorescence signal in inflamed mice, indicating endogenous HClO detection.
Conclusions:
- PMN-TPP is a highly sensitive and selective ratiometric fluorescent probe for mitochondrial ClO-.
- The probe is suitable for imaging endogenous HClO in both cellular and in vivo models of inflammation.
- PMN-TPP offers a valuable tool for studying the biological roles of hypochlorite.
