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Concept: Bioluminescence imaging


Bioluminescent imaging (BLI) has been widely applicable in the imaging of process envisioned in life sciences. As the most conventional technique for BLI, the firefly luciferin-luciferase system is exceptionally functional in vitro and in vivo. The state-of-the-art strategy in such a system is to cage the luciferin, in which free luciferin is conjugated with distinctive functional groups, thus accommodating an impressive toolkit for exploring various biological processes, such as monitoring enzymes activity, detecting bioactive small molecules, evaluating the properties of molecular transporters, etc. This review article summarizes the rational design of caged luciferins towards diverse biotargets, as well as their applications in bioluminescent imaging. It should be emphasized that these caged luciferins can stretch out the applications of bioluminescence imaging and shed light upon understanding the pathogenesis of various diseases.

Concepts: Fungus, In vivo, In vitro, Firefly, Bioluminescence, Luciferase, Luciferin, Bioluminescence imaging


As a trace element nutrient, cobalt is critical for both prokaryotes and eukaryotes. In the current study, a turn-on Cobalt Bioluminescent Probe 1 (CBP-1) for the detection of cobalt has been successfully developed based on oxidative C-O bond cleavage. This probe exhibited high selectivity and sensitivity towards cobalt over other analytes. By using CBP-1, the successful in vivo imaging of cobalt accumulation was carried out in a mouse model. Such an ability to determine cobalt in living animals provides a powerful technology for studying the system distribution, toxic potency, and biological effect of Co2+.

Concepts: Bacteria, Organism, Fungus, Animal, Model organism, Nitrogen, Bioluminescence, Bioluminescence imaging


Mercury is a highly toxic environmental pollutant that negatively affects human health. Thus, an in vivo method for noninvasive imaging of mercury(ii) and visualization of its accumulation within living systems would be advantageous. Herein, we describe a reaction-based bioluminescent probe for detection of mercury(ii) in vitro and accumulation in vivo. The application of this probe would help to shed light on the intricate contributions of mercury(ii) to various physiological and pathological processes.

Concepts: Health, Human, Light, In vivo, Toxicology, In vitro, Bioluminescence, Bioluminescence imaging


We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.

Concepts: Light, In vivo, Color, Red, Modification, Firefly, Bioluminescence, Bioluminescence imaging


Bioluminescence tomography is a preclinical imaging modality to locate and quantify internal bioluminescent sources from surface measurements, which experienced rapid growth in the last ten years. However, multiple-source resolving remains a challenging issue in BLT. In this paper, it is treated as an unsupervised pattern recognition problem based on the reconstruction result, and a novel hybrid clustering algorithm combining the advantages of affinity propagation (AP) and K-means is developed to identify multiple sources automatically. Moreover, we incorporate the clustering analysis into a general multiple-source reconstruction framework, which can provide stable reconstruction and accurate resolving result without providing the number of targets. Numerical simulations and in vivo experiments on 4T1-luc2 mouse model were conducted to assess the performance of the proposed method in multiple-source resolving. The encouraging results demonstrate significant effectiveness and potential of our method in preclinical BLT applications.

Concepts: Medical imaging, Model organism, Machine learning, Reconstruction era of the United States, Pattern recognition, Bioluminescence, Bioluminescence imaging, Hybrid word


Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 10(4) cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.

Concepts: Fluorescence, Cancer, Light, Quorum sensing, Firefly, Bioluminescence, Bioluminescence imaging, Omphalotus olearius


Bioluminescence tomography (BLT) can provide in vivo three-dimensional (3D) images for quantitative analysis of biological processes in preclinical small animal studies, which is superior than the conventional planar bioluminescence imaging. However, to reconstruct light sources under the skin in 3D with desirable accuracy and efficiency, BLT has to face the ill-posed and ill-conditioned inverse problem. In this paper, we developed a new method for BLT reconstruction, which utilized the mathematical strategies of the split Bregman iterative and surrogate functions (SBISF) method.

Concepts: Scientific method, Light, Medical imaging, Computer graphics, Bioluminescence, Inverse problem, Well-posed problem, Bioluminescence imaging


A novel bioluminescence probe for mercury(II) was obtained on the basis of the distinct deprotection reaction of dithioacetal to decanal, so as to display suitable sensitivity and selectivity toward mercury(II) over other ions with bacterial bioluminescence signal. These experimental results indicated such a probe was a novel promising method for mercury(II) bioluminescence imaging in environmental and life sciences ex vivo and in vivo.

Concepts: Gene, Bacteria, Fungus, Sensitivity and specificity, In vivo, Selectivity, Bioluminescence, Bioluminescence imaging


The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a non-ideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a non-biased manner, allowing the audience to adopt their own views of this novel system.

Concepts: Molecular biology, Enzyme, Quorum sensing, Field, Firefly, Bioluminescence, Luciferase, Bioluminescence imaging


Bioluminescence-based techniques, such as bioluminescence imaging, BRET and dual-luciferase reporter assay systems, have been widely used to examine a myriad of biological processes. Coelenterazine (CTZ), a luciferin or light-producing compound found in bioluminescent organisms, has sparked great curiosity and interest in searching for analogues with improved photochemical properties. This review summarizes the current development of coelenterazine analogues, their bioluminescence properties, and the rational design of caged coelenterazine towards biotargets, as well as their applications in bioassays. It should be emphasized that the design of caged luciferins can provide valuable insight into detailed molecular processes in organisms and will be a trend in the development of bioluminescent molecules.

Concepts: DNA, Molecule, Model organism, Chemistry, Chemical bond, Bioluminescence, Luciferin, Bioluminescence imaging