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Bioluminescence is a natural process where biological organisms produce light through chemical reactions. These reactions predominantly occur between small-molecule substrates and luciferase within bioluminescent organisms. Bioluminescence imaging (BLI) has shown significant potential in biomedical research owing to its non-invasive, real-time observation and quantification. In this review, we introduced the chemical mechanism of bioluminescent systems and categorized several strategies that successfully addressed the native limitations, including improvements on the chemical structures of luciferase-luciferin bioluminescence system and bioluminescence resonance energy transfer (BRET) methods. In addition, we also reviewed and summarized recent advances in bioimaging applications. We hope that this review can provide effective guidance for the development and application of bioluminescent systems in the field of bioimaging.

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Aflatoxin B1 (AFB1) is a potent carcinogen, and is among the most hazardous mycotoxins in agricultural products. Therefore, the development of sensitive and convenient detection methods for AFB1 is significant for food safety against mycotoxins. Herein, a bioluminescent enzyme immunoassay (BLEIA) was developed for ultrasensitive detection of AFB1, based on the novel Fc-specific antibody-nanoluciferase (Ab-Nluc) conjugates which were fabricated using an IgG-binding protein-assisted photo-conjugation strategy. In indirect competitive immunoassay format, the proposed BLEIA exhibited the detection limit of 0.0232 ng mL-1, which was 37.4-fold lower than that obtained using the classical enzyme-linked immunosorbent assay (ELISA) based on Ab-horseradish peroxidase (Ab-HRP) chemical conjugates (0.868 ng mL-1). Meanwhile, the BLEIA exhibited high accuracy and precision. Thus, the proposed Fc-specific Ab-Nluc conjugates-based BLEIA provides an ultrasensitive and reliable method for detecting toxins and has potential for use in food safety monitoring.

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Luminescent technology based on the luciferin-luciferase reaction has been extensively employed across various disciplines as a quantitative imaging modality. Owing to its non-invasive imaging capacity, it has evolved as a valuable in vivo bioimaging tool, particularly in small animal models in fields such as gene and cell therapies. We have previously successfully generated rats with a systemic expression of the luciferase gene at the Rosa26 locus. In this study, we transplanted bone marrow from these rats into micro-mini pigs and used in vivo imaging to non-invasively analyze the dynamics of the transplanted cells. In addition, we established that the rat-to-pig transplantation system is a discordant system, similar to the pig-to-human transplantation system. Thus, rat-to-pig transplantation may provide a clinically appropriate large animal model for pig-to-human xenotransplantation.

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Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector's ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2's ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases.

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Staphylococcus aureus [S. aureus] is a leading cause of sepsis and septic arthritis, conditions that pose significant medical challenges due to their high mortality and morbidity. No studies have used an in vivo imaging system [IVIS] to monitor S. aureus sepsis and septic arthritis. Here, we employed a bioluminescent reporter strain of S. aureus, Newman AH5016, administered intravenously to induce sepsis and intra-articularly to induce local septic arthritis in mice. Disease progression was monitored using IVIS to capture bioluminescent signals from kidneys, joints, and whole mice. Cytokines in the blood and joints were measured. The efficacy of cloxacillin treatment was evaluated. In the sepsis model, bioluminescent signals from kidneys, but not from whole mice, were correlated with kidney bacterial load and abscess formation. Ex vivo kidney imaging detected increased bacterial load and abscess formation from day 3 to day 10. Antibiotic treatment significantly reduced kidney signals, correlating with decreased bacterial counts and IL-6 levels, indicating effective infection control. In the local infection model, early-phase bioluminescent signals from joints were correlated with macroscopic arthritis and bacterial burden. Thus, signal detection from kidneys using IVIS is useful for monitoring S. aureus sepsis and assessing antibiotic efficacy, though it may only be effective for early-phase monitoring of local septic arthritis.

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Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin-luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.

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Significance: The choroid plexus (ChP) epithelial network displays diverse dynamics, including propagating calcium waves and individuated fluctuations in single cells. These rapid events underscore the possibility that ChP dynamics may reflect behaviorally relevant and clinically important changes in information processing and signaling. Optogenetic and chemogenetic tools provide spatiotemporally precise and sustained approaches for testing such dynamics in vivo. Here, we describe the feasibility of a novel combined opto- and chemogenetic tool, BioLuminescent-OptoGenetics (BL-OG), for the ChP in vivo. In the "LuMinOpsin" (LMO) BL-OG strategy, a luciferase is tethered to an adjacent optogenetic element. This molecule allows chemogenetic activation when the opsin is driven by light produced through luciferase binding a small molecule (luciferin) or by conventional optogenetic light sources and BL-OG report of activation through light production. Aim: To test the viability of BL-OG/LMO for ChP control. Approach: Using transgenic and Cre-directed targeting to the ChP, we expressed LMO3 (a Gaussia luciferase-VChR1 fusion), a highly effective construct in neural systems. In mice expressing LMO3 in ChP, we directly imaged BL light production following multiple routes of coelenterazine (CTZ: luciferin) administration using an implanted cannula system. We also used home-cage videography with Deep LabCut analysis to test for any impact of repeated CTZ administration on basic health and behavioral indices. Results: Multiple routes of CTZ administration drove BL photon production, including intracerebroventricular, intravenous, and intraperitoneal injection. Intravenous administration resulted in fast "flash" kinetics that diminished in seconds to minutes, and intraperitoneal administration resulted in slow rising activity that sustained hours. Mice showed no consistent impact of 1 week of intraperitoneal CTZ administration on weight, drinking, motor behavior, or sleep/wake cycles. Conclusions: BL-OG/LMO provides unique advantages for testing the role of ChP dynamics in biological processes.

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Currently, risk assessment and pollution management in mines primarily focus on toxic metals, with the flotation agents being overlooked. However, the combined effects of metals and flotation agents in mines remain largely unknown. Therefore, this study aimed to evaluate the combined effects of Cd and two organic flotation agents (ethyl xanthate (EX) and diethyldithiocarbamate (DDTC)), and the associated mechanisms. The results showed that Cd + EX and Cd + DDTC exhibited synergistic toxicity. The EC50 values for luminescent bacteria were 1.6 mg/L and 1.0 mg/L at toxicity unit ratios of 0.3 and 1, respectively. The synergistic effects were closely related with the formation of Cd(EX)2 and Cd(DDTC)2 micro/nano particles, with nano-particles exhibiting higher toxicity. We observed severe cell membrane damage and cell shrinkage of the luminescent bacteria, which were probably caused by secondary harm to cells through the released CS2 during their decomposition inside cells. In addition, these particles induced toxicity by altering cellular levels of biochemical markers and the transcriptional levels of transport proteins and lipoproteins, leading to cell membrane impairment and DNA damage. This study has demonstrated that particulates formed by Cd and flotation agents contribute to the majority of the toxicity of the binary mixture. This study helps to better understand the complex ecological risk of inorganic metals and organic flotation agents in realistic mining environments.

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Bioluminescence imaging (BLI) is a powerful method for visualizing biological processes and tracking cells. Engineered bioluminescent bacteria that utilize luciferase-catalyzed biochemical reactions to generate luminescence have become useful analytical tools for in vitro and in vivo bacterial imaging. Accordingly, this review initially introduces the development of engineered bioluminescent bacteria that use different luciferase-luciferin pairs as analytical tools and their applications for in vivo BLI, including real-time bacterial tracking of infection, probiotic investigation, tumor-targeted therapy, and drug screening. Applications of engineered bioluminescent bacteria as whole-cell biosensors for sensing biological changes in vitro and in vivo are then discussed. Finally, we review the optimizations and future directions of bioluminescent bacteria for imaging. This review aims to provide fundamental insights into bacterial BLI and highlight the potential development of this technique in the future.

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Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.

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This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

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Significance: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions: We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

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Aminopeptidases, enzymes with critical roles in human body, are emerging as vital biomarkers for metabolic processes and diseases. Aberrant aminopeptidase levels are often associated with diseases, particularly cancer. Small-molecule probes, such as fluorescent, fluorescent/photoacoustics, bioluminescent, and chemiluminescent probes, are essential tools in the study of aminopeptidases-related diseases. The fluorescent probes provide real-time insights into protein activities, offering high sensitivity in specific locations, and precise spatiotemporal results. Additionally, photoacoustic probes offer signals that are able to penetrate deeper tissues. Bioluminescent and chemiluminescent probes can enhance in vivo imaging abilities by reducing the background. This comprehensive review is focused on small-molecule probes that respond to four key aminopeptidases: aminopeptidase N, leucine aminopeptidase, Pyroglutamate aminopeptidase 1, and Prolyl Aminopeptidase, and their utilization in imaging tumors and afflicted regions. In this review, the design strategy of small-molecule probes, the variety of designs from previous studies, and the opportunities of future bioimaging applications are discussed, serving as a roadmap for future research, sparking innovations in aminopeptidase-responsive probe development, and enhancing our understanding of these enzymes in disease diagnostics and treatment.

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Staphylococcus aureus nasal carriage is considered a risk factor for infections, and the development of nasal decolonization strategies is highly relevant. Despite they are not naturally colonized by Staphylococcus, mice are a good model for S. aureus nasal colonization. Murine models are easy to manipulate, and inter-laboratory reproducibility makes them suitable for nasal colonization studies. Strategies using bioluminescent bacteria allow for the monitoring of infection over time without the need to sacrifice animals for bacterial quantification. In this study, we evaluated S. aureus nasal colonization in three mouse strains (BALB/c, C57BL/6, and Swiss Webster) using a bioluminescent strain (SAP231). In vitro, a visible Bioluminescent Signal Emission (BLSE) was observed until 106 bacteria and detected by IVIS® imaging system up to 104 cells. Animals were inoculated with one or two doses of approximately 109 colony-forming units (CFU) of SAP231. Swiss Webster mice showed the longest colonization time, with some animals presenting BLSE for up to 140 h. In addition, BLSE was higher in this strain. BALB/c and C57BL/6 strains showed consistent BLSE results for 48 h. BLSE intensity was higher in Swiss Webster inoculated with both doses. Three different positions for image capture were evaluated, with better results for the lateral and ventrodorsal positions. After the loss of BLSE, bacterial quantification was performed, and Swiss Webster mice presented more bacteria in the nasal cavity (approximately 105 CFU) than the other strains. Our results demonstrate that bioluminescent S. aureus allow monitoring of nasal colonization and estimation of the bacterial burden present in live animals until 48 h.

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Orthotopic models of hepatocellular carcinoma (HCC) consist in the implantation of tumor cells into the liver by direct intrahepatic injection. In this model, tumorigenesis is triggered within the hepatic microenvironment, thus mimicking the metastatic behavior of HCC. Herein, we detail a surgically mediated methodology that allows the reproducible and effective induction of liver-sessile tumors in mice. We enumerate the steps to be followed before and after the surgical procedure, including HCC cell preparation, the quantity of cancer cells to be injected, presurgical preparation of the mice, and finally, postoperative care. The surgical procedure involves laparotomy to expose the liver, injection of cells into the left-lateral hepatic lobe, and closure of the incision with sutures followed by wound clips. We also provide information concerning the subsequent tumor growth follow-up, as well as the application of bioluminescence imaging to monitor tumor development.

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Interest in enzymes capable of neutralizing various mycotoxins is quite high. The methods used for the screening and selection of enzymes that catalyze the detoxification of mycotoxins should be sensitive and fast. However toxic compounds can be generated under the action of such enzymes. Thus, the assessment of the overall reduction in the toxic properties of reaction media towards bioluminescent bacteria seems to be the most reasonable control method allowing a quick search for the effective enzymatic biocatalysts. The influence of a wide range of mycotoxins and glucanases, which hydrolyze toxins with different chemical structures, on the analytical characteristics of luminescent photobacteria as a biosensing element has been studied. Different glucanases (ß-glucosidase and endoglucanase) were initially selected for reactions with 10 mycotoxins based on the results of molecular docking which was performed in silico with 20 mycotoxins. Finally, the biorecognizing luminescent cells were used to estimate the residual toxicity of reaction media with mycotoxins after their interaction with enzymes. The notable non-catalytic decrease in toxicity of media containing deoxynivalenol was revealed with luminous cells for both types of tested glucanases, whereas ß-glucosidase provided a significant catalytic detoxification of media with aflatoxin B2 and zearalenone at pH 6.0.

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Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

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Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic nematodes. As part of our ongoing exploration to identify bioactive compounds from diverse natural resources, the chemical analysis of the cultures of P. luminescens KACC 12254 via LC/MS and TLC-based analyses enabled the isolation and identification of a major fluorescent compound. Its chemical structure was elucidated as 1,8-dihydroxy-3-methoxyanthraquinone (DMA) using HR-ESI-MS and NMR analysis. In this study, we conducted comprehensive investigations utilizing human colorectal cancer HCT116 cells, human umbilical cord vascular endothelial cells (HUVECs), and zebrafish embryos to assess the potential benefits of DMA in suppressing tumor angiogenesis. Our results convincingly demonstrate that DMA effectively suppresses the stability of hypoxia-inducible factor-1α (HIF-1α) protein and its target genes without inducing any cytotoxic effects. Furthermore, DMA demonstrates the ability to inhibit HIF-1α transcriptional activation and mitigate the production of reactive oxygen species (ROS). In our in vitro experiments, DMA exhibits notable inhibitory effects on VEGF-mediated tube formation, migration, and invasion in HUVECs. Additionally, in vivo investigations using zebrafish embryos confirm the antiangiogenic properties of DMA. Notably, DMA does not exhibit any adverse developmental or cardiotoxic effects in the in vivo setting. Moreover, we observe DMA's capability to restrain tumor growth through the downregulation of PI3K/AKT and c-RAF/ERK pathway. Collectively, these compelling findings underscore DMA's potential as a promising therapeutic candidate for targeted intervention against HIF-1α and angiogenesis in cancer treatment.

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Background: Colorectal cancer (CRC) is the fifth most fatal cancer with a low probability of surgery and limited treatment options, especially in metastatic CRC. In this study, we investigated whether a mouse model of metastatic CRC mimicked tumor progression and evaluated the effect of 5-fluorouracil (5-FU) treatment. Methods: The CT26 mouse derived CRC cancer cell line was inoculated into mice, and the tumor bearing mice were divided into two groups: the experimental group and the control group. Micro-computed tomography (CT) and in vivo fluorescence were used to monitor the progression of metastatic CRC. A lung metastasis mouse model was employed to determine the effects of 5-FU on metastasis. Results: Bioluminescence imaging (BLI) and computed tomography (CT), as non-invasive methods, can continuously monitor the growth of tumors in vivo. Thus, imaging techniques can be used to qualitatively and quantitatively evaluate tumor growth indicators. 5-FU injected intravenously reduced the viability of metastatic CRC cells and resulted in prolonged survival compared to the control group. Moreover, the 5-FU-treated group had significantly reduced fluorescence of the CT26 cells in the lung. The results observed by BLI and CT are consistent with the tissue morphology and structure presented in pathological examination. Conclusions: In summary, a successful mouse model of CRC metastasis for clinical application has been established.

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Type I interferon-beta (IFN-ß) is a crucial component of innate and adaptive immune systems inside the host. The formation of bacterial biofilms on medical implants can lead to inflammatory diseases and implant failure. Biofilms elicit IFN-ß production inside the host that, in turn, restrict bacterial growth. Biofilms pose strong antibiotic resistance, whereas surface modification of medical implants with antibacterial agents may demonstrate strong antimicrobial effects. Most of the previous investigations were focused on determining the antibacterial activities of implant surfaces modified with antibacterial agents. The present study, for the first time, measured antibacterial activities and IFN-ß expression of titanium surfaces along with silver or tetracycline inside co-culture and mouse models. A periodontal pathogen: Aggregatibacter actinomycetemcomitans reported to induce strong inflammation, was used for infection. Silver and tetracycline were added to the titanium surface using the heat evaporation method. Macrophages showed reduced compatibility on titanium surfaces with silver, and IFN-ß expression inside cultured cells significantly decreased. Macrophages showed compatibility on implant surfaces with tetracycline, but IFN-ß production significantly decreased inside seeded cells. The decrease in IFN-ß production inside macrophages cultured on implant surfaces with silver and tetracycline was not related to the downregulation of Ifn-ß gene. Bacterial infection significantly upregulated mRNA expression levels of Isg15, Mx1, Mx2, Irf-3, Irf-7, Tlr-2, Tnf-α, Cxcl-1, and Il-6 genes. Notably, mRNA expression levels of Mx1, Irf7, Tlr2, Tnf-α, Cxcl1, and Il-6 genes inside macrophages significantly downregulated on implant surfaces with silver or tetracycline. Titanium with tetracycline showed higher antibacterial activities than silver. The in vivo evaluation of IFN-ß expression around implants was measured inside transgenic mice constitutive for IFN-ß expression. Of note, the non-invasive in vivo imaging revealed a significant decrease in IFN-ß expression around subcutaneous implants with silver compared to titanium and titanium with tetracycline in sterile or infected situations. The histology of peri-implant tissue interfaces around infected implants with silver showed a thick interface with a significantly higher accumulation of inflammatory cells. Titanium implants with silver and tetracycline remained antibacterial in mice. Findings from this study unequivocally indicate that implant surfaces with silver decrease IFN-ß expression, a crucial component of host immunity.