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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.

Keywords:
HaloTagcell imagingchemigenetic probesfluorescencefluorescence lifetime imagingprotein structure prediction

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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.