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Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Published on: January 24, 2017
Polarity-responsive probe for dual-channel visualization of mitochondrial membrane potential via subcellular
Qilong Zhang1, Bingbing Pan2, Ranxu Zhou2
1School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, China; TongChuang Chemical (ShanDong) CO., LTD, Jining, Shandong 272600, PR China.
Abstract:
Mitochondrial membrane potential (∆Ψm) plays central roles in cell apoptosis, signalling, metabolism, and other crucial bioevents. However, most of the current dual-emissive fluorescent probes for ∆Ψm works on aggregation mechanism, rendering possible interferences from inhomogeneous staining, cell numbers, and other factors. To resolve this knot, in this work we have developed a dual-emissive fluorescent probe detecting ∆Ψm based on polarity-response mechanism. The probe was designed to display different emission wavelength under different polarity, and simultaneously high affinity to both mitochondria and RNA. In live cells, mitochondria maintain a high negative transmembrane potential. Under this condition, the probe specifically targeted mitochondria because of its cationic structure. After the depolarization of ∆Ψm, the probe lost its mitochondrial targeting capability and shifted the target to RNA owing to its high RNA affinity. The distinct polar environments between the mitochondrial inner membrane and RNA result in a significant red shift in the fluorescence wavelength of the probe after its relocation from mitochondria to RNA. In this manner, the probe enabled the dual-emissive evaluation of the ∆Ψm. The probe was successfully applied to visualize the reversible change of ∆Ψm in real-time and in-situ manner. With the probe, the decrease of ∆Ψm in apoptosis procedure induced by toxins and anti-tumor drugs was also detected.
Insights
This study introduces a novel dual-emissive fluorescent probe for monitoring mitochondrial membrane potential (∆Ψm). The probe utilizes a polarity-response mechanism, offering a more reliable method for studying cell apoptosis and metabolism.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Probes
Background:
- Mitochondrial membrane potential (∆Ψm) is critical for cellular processes like apoptosis, signaling, and metabolism.
- Existing dual-emissive probes for ∆Ψm often rely on aggregation mechanisms, leading to potential inaccuracies due to staining variability and cell density.
- A need exists for more robust and reliable probes to accurately assess ∆Ψm.
Purpose of the Study:
- To develop a novel dual-emissive fluorescent probe for ∆Ψm detection based on a polarity-response mechanism.
- To overcome the limitations of aggregation-based probes by designing a probe with distinct emission characteristics in different cellular environments.
- To enable real-time and in-situ visualization of reversible ∆Ψm changes and its role in apoptosis.
Main Methods:
- Designed a dual-emissive fluorescent probe with a cationic structure for mitochondrial targeting and high RNA affinity.
- Exploited the probe's differential emission wavelength shifts in response to polarity changes between mitochondrial membranes and RNA.
- Applied the probe in live cells to visualize ∆Ψm dynamics and detect changes during apoptosis induced by toxins and anti-tumor drugs.
Main Results:
- The developed probe successfully targets mitochondria in live cells with high membrane potential.
- Upon mitochondrial depolarization, the probe relocates to RNA, exhibiting a significant red shift in fluorescence emission due to polarity differences.
- The probe enabled real-time, in-situ visualization of reversible ∆Ψm changes and detected decreased ∆Ψm during drug-induced apoptosis.
Conclusions:
- A novel polarity-response based dual-emissive fluorescent probe for ∆Ψm has been successfully developed.
- This probe overcomes limitations of aggregation-based methods, offering accurate and reliable ∆Ψm monitoring.
- The probe is effective for visualizing dynamic ∆Ψm alterations in cellular processes, including apoptosis.
