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Introduction
In vivo molecular imaging techniques that enable in situ visualization and longitudinal monitoring biological processes in live animals are indispensable for both basic research and biomedical applications. Among them, bioluminescence imaging (BLI) stands out as a powerful technique with unique advantages1, 2–3. As an optical method, BLI has become widely used in preclinical research, by enabling non-invasive imaging of genetically specified cell populations or molecular events at low cost. BLI provides a distinct combination of benefits including good sensitivity and resolution for real-time tracking molecular events, relatively low operational costs and the ability to perform longitudinal repeated imaging studies without radiation risk4. While other biomedical imaging modalities offer distinct strengths—such as the superior anatomical resolution of MRI, the high sensitivity of PET for tracking radiolabeled molecules, and the desirable temporal resolution and low costs of ultrasound—BLI has established itself as an important complementary technique in the preclinical imaging toolbox. In addition, developments in genetically encodable bioluminescent indicators introduce the ability to track various biochemical events in vivo5,6. Compared to fluorescence imaging, another widely-used optical imaging method, BLI eliminates the need for excitation light, which generates autofluorescent background and suffers attenuation with depth. Consequently, BLI achieves superior signal-to-background ratios at deeper locations. Overall, BLI allows non-invasive studies of gene expression, cell localization, and molecular events in living animal models. Its suitability for long-term, longitudinal studies with minimal disturbance makes it widely used in small mammalian disease models for studying disease progression and evaluating treatments1, 2, 3, 4, 5–6.
Two classes of bioluminescent enzyme–substrate systems have been widely adopted in preclinical imaging: insect luciferases that use D-luciferin and related compounds, O2, and ATP as substrates (such as firefly luciferases, FLuc) and marine luciferases that use coelenterazine and related compounds and O2 as substrates (such as Renilla luciferase, RLuc)1, 2–3. FLuc-based BLI has gained popularity due to its emission peak in the yellow to red spectral region (≥ 560 nm), which enables superior tissue penetration compared to shorter-wavelength luciferases, and the luciferin’s water solubility and in vivo bioavailability. However, insect luciferases require ATP, which prevents their use in extracellular compartments or for tracking biochemical events when ATP levels fluctuate7. In contrast, marine...