Semisynthetic, Multicolor Probes for Bioluminescence Imaging

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.