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Engineered HaloTag variants for fluorescence lifetime multiplexing.

Michelle S Frei1,2, Miroslaw Tarnawski3, M Julia Roberti4

  • 1Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.

Nature Methods
|December 17, 2021
PubMed
Summary
This summary is machine-generated.

New HaloTag variants offer enhanced brightness and tunable fluorescence lifetimes for advanced live-cell imaging. This enables multiplexing of cellular targets and biosensor development using a single fluorophore.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Microscopy

Background:

  • Self-labeling protein tags like HaloTag are crucial for fluorescent labeling of proteins in microscopy.
  • Existing tags have limitations in brightness and multiplexing capabilities.

Purpose of the Study:

  • To develop novel HaloTag variants with modulated brightness and fluorescence lifetime.
  • To enable advanced live-cell imaging techniques, including multiplexing and biosensing.

Main Methods:

  • Engineering of HaloTag variants with altered photophysical properties.
  • Labeling with rhodamine fluorophores.
  • Live-cell fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM).

Main Results:

  • Developed HaloTag variants with increased and decreased brightness and fluorescence lifetime compared to HaloTag7.
  • Achieved live-cell fluorescence lifetime multiplexing of three targets in a single spectral channel.
  • Demonstrated a fluorescence lifetime-based biosensor.
  • The brightest variant exhibited up to 40% increased brightness in live-cell imaging.

Conclusions:

  • Novel HaloTag variants significantly enhance live-cell fluorescence imaging capabilities.
  • These variants facilitate advanced multiplexing and biosensor applications.
  • The developed tools offer improved performance for fluorescence microscopy.