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Related Experiment Video

Updated: Jan 8, 2026

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL
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Researchers developed a novel bioluminescence imaging method using NanoBiT, a split luciferase, to overcome NanoLuc

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Bioluminescence imaging offers sensitive, noninvasive monitoring of biological processes.
  • NanoLuc luciferase provides bright signals but its blue emission limits tissue penetration.
  • NanoBiT, a split NanoLuc system, presents an opportunity for modified emission properties.

Purpose of the Study:

  • To red-shift the emission of NanoBiT for improved bioluminescence imaging applications.
  • To develop a versatile platform for multicolor bioluminescence imaging.
  • To enhance sample penetration and enable new imaging modalities.

Main Methods:

  • Chemically labeling the HiBiT subunit of NanoBiT with fluorescent probes.
  • Utilizing bioluminescence resonance energy transfer (BRET) to transfer energy from NanoBiT to attached fluorophores.
  • Conjugating HiBiT peptides to fluorescent quantum dots for near-infrared emission.
  • Employing spectral phasor analysis microscopy to distinguish emission profiles.

Main Results:

  • Successful red-shifting of NanoBiT emission by chemically attaching fluorescent probes.
  • Generation of unique emission profiles for multicolor imaging using spectral phasor analysis.
  • Extension of BRET emission into the near-infrared spectrum by conjugating HiBiT to quantum dots.
  • Demonstration of a modular system with tunable emission from visible to near-infrared.

Conclusions:

  • The developed NanoBiT-based system provides a versatile platform for multicolor bioluminescence imaging.
  • This approach overcomes the limitations of blue-shifted bioluminescence, enabling deeper tissue penetration.
  • The modularity and tunability offer significant advantages for various biological imaging applications.