An optimized bioluminescent substrate for non-invasive imaging in the brain

Area of Science:

• Neuroscience
• Biomedical Imaging
• Molecular Biology

Background:

• Bioluminescence imaging (BLI) is crucial for in vivo research but faces limitations in the central nervous system due to poor luciferase performance.
• Existing substrates and luciferases provide insufficient signal for effective brain imaging.

Purpose of the Study:

• To discover and characterize a novel NanoLuc substrate with enhanced performance for bioluminescence imaging in the brain.
• To enable sensitive, non-invasive visualization of biological processes within the central nervous system.

Main Methods:

• Development and testing of a new NanoLuc substrate, cephalofurimazine (CFz).
• Comparison of CFz/Antares luciferase system with standard D-luciferin/firefly luciferase and AkaLuc/AkaLumine systems.
• In vivo imaging in freely moving mice, including video-rate imaging and calcium imaging of neuronal activity.

Main Results:

• Cephalofurimazine (CFz) with Antares luciferase yields over 20-fold greater signal in the brain compared to D-luciferin/firefly luciferase.
• The Antares-CFz combination demonstrates comparable brightness to AkaLuc-AkaLumine/TokeOni at standard doses.
• Higher doses of CFz can achieve up to threefold signal enhancement.
• Successful video-rate non-invasive imaging of Antares in mouse brains and calcium imaging of sensory-evoked neuronal activity were achieved.

Conclusions:

• Cephalofurimazine (CFz) represents a significant advancement for NanoLuc-based bioluminescence imaging in the brain.
• The improved sensitivity of CFz opens new possibilities for applying NanoLuc indicators to neuroscience research.
• This substrate facilitates high-resolution, non-invasive monitoring of neural dynamics in vivo.