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Fluorescence Lifetime Multiplexing with Environment-Sensitive Chemigenetic Probes
Sarah Emmert1, Anna Rovira1, Pablo Rivera-Fuentes1
1Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland.
Chembiochem : a European Journal of Chemical Biology
|April 30, 2025
Summary
This study explores using an environment-sensitive dye with HaloTag (HT) mutants for multiplexed fluorescence lifetime imaging microscopy (FLIM). This approach enables three-channel imaging by leveraging dye localization and efficient HT labeling.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- HaloTag (HT) is a widely used self-labeling protein in fluorescence microscopy.
- Multiplexed fluorescence lifetime imaging microscopy (FLIM) offers advanced imaging capabilities.
- Environment-sensitive fluorophores are key for advanced microscopy techniques.
Purpose of the Study:
- To explore the application of an environment-sensitive fluorophore for multiplexed FLIM with HaloTag (HT) mutants.
- To demonstrate three-channel imaging using a novel fluorophore and HT system.
- To investigate the influence of protein binding pocket polarity on fluorescent molecule lifetime.
Main Methods:
- Utilized an environment-sensitive extended coumarin pyridinium scaffold fluorophore.
- Employed two distinct HaloTag (HT) mutants for selective labeling.
- Investigated dye accumulation in cellular compartments like mitochondria and vesicles.
- Examined FLIM multiplexing capabilities with various dehalogenase proteins and HT mutants.
Main Results:
- Achieved three-channel imaging by combining free dye localization and HT-specific labeling.
- Demonstrated the environment-sensitive dye's utility in multiplexed FLIM.
- Identified protein binding pocket polarity as a critical factor influencing fluorescent molecule lifetime.
Conclusions:
- The developed fluorophore-HT system is effective for multiplexed FLIM.
- Cellular localization and protein interactions significantly impact fluorescence lifetime.
- This method advances multiplexed imaging in cell biology and biophysics.
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