Inspired by nature: Bioluminescent systems for bioimaging applications.

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.

Strength of activation and temporal dynamics of bioluminescent-optogenetics in response to systemic injections of the luciferin.

BioLuminescent OptoGenetics ("BL-OG") is a chemogenetic method that can evoke optogenetic reactions in the brain non-invasively. In BL-OG, an enzyme that catalyzes a light producing reaction (i.e., a luciferase) is tethered to an optogenetic element that is activated in response to bioluminescent light. Bioluminescence is generated by injecting a chemical substrate (luciferin, e.g., h-Coelenterazine; h-CTZ) that is catalyzed by the luciferase. By directly injecting the luciferin into the brain, we show that bioluminescent light is proportional to spiking activity, and this relationship scales as a function of luciferin dosage. Here, we build on these previous observations by characterizing the temporal dynamics and dose response curves of bioluminescence generated by luminopsins (LMOs), a proxy of BL-OG effects, to intravenous (IV) injections of the luciferin. We imaged bioluminescence through a thinned skull of mice running on a wheel, while delivering h-CTZ via the tail vein with different dosage concentrations and injection rates. The data reveal a systematic relationship between strength of bioluminescence and h-CTZ dosage, with higher concentration generating stronger bioluminescence. We also found that bioluminescent activity occurs rapidly (< 60 s after IV injection) regardless of concentration dosage. However, as expected, the onset time of bioluminescence is delayed as the injection rate decreases. Notably, the strength and time decay of bioluminescence is invariant to the injection rate of h-CTZ. Taken together, these data show that BL-OG effects are highly consistent across injection parameters of h-CTZ, highlighting the reliability of BL-OG as a minimally invasive neuromodulation method.

Shining new light on naupliar eyes: A novel molecular phylogeny for Pleuromamma (Family: Metridinidae) and the characterization of luciferase and opsin expression.

Pleuromamma (Giesbrecht, 1898) is a cosmopolitan genus of metridinid copepods, with species that perform remarkable diel vertical migrations (DVM) and emit a bioluminescent secretion when disturbed that varies both spectrally and kinetically. Copepod bioluminescence is autogenic and uses luciferase enzymes that catalyze a luciferin, coelenterazine, to produce light. Pleuromamma possess naupliar eyes, relatively simple photosensitive structures used for many visually-guided behaviors. Yet the fundamental molecular unit for vision, the opsin protein, has not been previously described for the family. The light producers and detectors are important to study because DVM is a behavior that mediates significant active elemental fluxes between the upper ocean and midwaters across vast stretches of oceanic habitat, and DVM is guided by visual behaviors, with animals tracking an isolume. Here we provide the first fully resolved molecular phylogeny for Pleuromamma (Family: Metridinidae) and describe the luciferase and opsin gene diversity and expression using de novo assembled transcriptomes. We successfully sequenced and assembled transcriptomes for 10 of 11 described species of Pleuromamma as well as two other metridinid species: Metridia longa and Gaussia princeps. In all species, we obtained coding sequences of one putative rhabdomeric middle wavelength sensitive visual opsin gene, as well as several non-visual opsins - a c-type pteropsin and a tetra-opsin type peropsin. Furthermore, Pleuromamma express luciferases from each of two main evolutionary clades (Luc1 and Luc2), and a single paralog (Luc2a) dominates expression throughout the group. The variation in luciferase number, sequence, and expression among species could lead to different spectral and kinetic properties of bioluminescence and aid in congener recognition.

New transaminase from Odontosyllis undecimdonta the first potential enzyme of the luciferin biosynthesis pathway.

Among nearly a hundred known bioluminescent systems, only about a dozen have been studied to some extent, and the structures of only a few luciferins have been established. Moreover, the biosynthesis pathway is known only for two of them - the fungal and bacterial ones. Marine polychaetes of the Odontosyllis genus possess bright bioluminescence. The structures of its bioluminescence system key components were recently elucidated, and a possible pathway of the luciferin biosynthesis was proposed. Here we report the transaminase enzyme from Odontosyllis undecimdonta, the first potential participant of the cascade. We demonstrate that the discovered ferment catalyzes the transamination of the cys2DOPA, one of the potential luciferin biosynthetic precursors. The results of the experiments support the hypothesis that the discovered enzyme might be the part of the Odontosyllis luciferin biosynthesis pathway.

Non-Invasive Direct Visualization of Luciferase-Luciferin Emitted Light Producing True Images from an Orthotopic Lung Cancer Xenograft Model in Nude Mice Using an Ultra-sensitive Low-light Camera and Optics.

BACKGROUND/AIM: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. RESULTS: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. CONCLUSION: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal.

A novel method for measurements of sleep/wake states, feeding and drinking behaviors using the tracking technique of 3D positions in freely moving mice.

We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

Unimolecular Chemiexcited Oxygenation of Pathogenic Amyloids.

Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-ß (Aß), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aß amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.

The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in in vitro susceptibility testing of Madurella mycetomatis.

BACKGROUND: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. METHODS: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. RESULTS: Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC. CONCLUSION: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. LAY SUMMARY: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them.

A reproducible and affordable method of conducting luciferase assay.

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

Advances in the Development of Bacterial Bioluminescence Imaging.

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.

Bioluminescence of (R)-Cypridina Luciferin with Cypridina Luciferase.

Cypridina luciferin (CypL) is a marine natural product that functions as the luminous substrate for the enzyme Cypridina luciferase (CypLase). CypL has two enantiomers, (R)- and (S)-CypL, due to its one chiral center at the sec-butyl moiety. Previous studies reported that (S)-CypL or racemic CypL with CypLase produced light, but the luminescence of (R)-CypL with CypLase has not been investigated. Here, we examined the luminescence of (R)-CypL, which had undergone chiral separation from the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that (R)-CypL with CypLase produced light, indicating that (R)-CypL must be considered as the luminous substrate for CypLase, as in the case of (S)-CypL, rather than a competitive inhibitor for CypLase. Additionally, we found that the maximum luminescence intensity from the reaction of (R)-CypL with CypLase was approximately 10 fold lower than that of (S)-CypL with CypLase, but our kinetic analysis of CypLase showed that the Km value of CypLase for (R)-CypL was approximately 3 fold lower than that for (S)-CypL. Furthermore, the chiral high-performance liquid chromatography (HPLC) analysis of the reaction mixture of racemic CypL with CypLase showed that (R)-CypL was consumed more slowly than (S)-CypL. These results indicate that the turnover rate of CypLase for (R)-CypL was lower than that for (S)-CypL, which caused the less efficient luminescence of (R)-CypL with CypLase.

Recent advances in bioluminescent probes for neurobiology.

Bioluminescence is a popular modality for imaging in living organisms. The platform relies on enzymatically (luciferase) generated light via the oxidation of small molecule luciferins. Since no external light is needed for photon production, there are no concerns with background autofluorescence or photobleaching over time-features that have historically limited other optical readouts. Bioluminescence is thus routinely used for longitudinal tracking across whole animals. Applications in the brain, though, have been more challenging due to a lack of sufficiently bioavailable, bright, and easily multiplexed probes. Recent years have seen the development of designer luciferase and luciferin pairs that address these issues, providing more sensitive and real-time readouts of biochemical features relevant to neurobiology. This review highlights many of the advances in bioluminescent probe design, with a focus on the small molecule light emitter, the luciferin. Specific efforts to improve luciferin pharmacokinetics and tissue-penetrant emission are covered, in addition to applications that such probes have enabled. The continued development of improved bioluminescent probes will aid in illuminating critical neurochemical processes in the brain.

Validation and application of caged Z-DEVD-aminoluciferin bioluminescence for assessment of apoptosis of wild type and TLR2-deficient mice after ischemic stroke.

Programmed cell death or apoptosis is a critically important mechanism of tissue remodeling and regulates conditions such as cancer, neurodegeneration or stroke. The aim of this research article was to assess the caged Z-DEVD-aminoluciferin substrate for in vivo monitoring of apoptosis after ischemic stroke in TLR2-deficient mice and their TLR2-expressing counterparts. Postischemic inflammation is a significant contributor to ischemic injury development and apoptosis, and it is modified by the TLR2 receptor. Caged Z-DEVD-aminoluciferin is made available for bioluminescence enzymatic reaction by cleavage with activated caspase-3, and therefore it is assumed to be capable of reporting and measuring apoptosis. Apoptosis was investigated for 28 days after stroke in mice which ubiquitously expressed the firefly luciferase transgene. Middle cerebral artery occlusion was performed to achieve ischemic injury, which was followed with magnetic resonance imaging. The scope of apoptosis was determined by bioluminescence with caged Z-DEVD-aminoluciferin, immunofluorescence with activated caspase-3, flow cytometry with annexin-V and TUNEL assay. The linearity of Z-DEVD-aminoluciferin substrate dose effect was shown in the murine brain. Z-DEVD-aminoluciferin was validated as a good tool for monitoring apoptosis following adequate adjustment. By utilizing bioluminescence of Z-DEVD-aminoluciferin after ischemic stroke it was shown that TLR2-deficient mice had lower post-stroke apoptosis than TLR2-expressing wild type mice. In conclusion, Z-DEVD-aminoluciferin could be a valuable tool for apoptosis measurement in living mice.

Constitutive and evoked release of ATP in adult mouse olfactory epithelium.

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin-luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.

The role of protein globule in firefly luciferase catalysis.

The important role of the dynamic structure of firefly luciferase in enzyme functioning is a subject of this literature review. Due to the domain alternation, the optimal configuration of the active site is created for each stage of the luciferin oxidation. The diversity of bioluminescence spectra is explained by the combined emission of several coexisting forms of electronically excited oxyluciferin. The superposition of two or three emitter forms recorded in the bioluminescence spectra indicates that different luciferase conformers coexist in the reaction medium in dynamic equilibrium. The relationship between the thermal stability of the protein globule and the bioluminescence spectra is also discussed.

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

Synthesis and Evaluation of Glycosyl Luciferins.

Measuring glycosidase activity is important to monitor any aberrations in carbohydrate hydrolase activity, but also for the screening of potential glycosidase inhibitors. To this end, synthetic substrates are needed which provide an enzyme-dependent read-out upon hydrolysis by the glycosidase. Herein, we present two new routes for the synthesis of caged luminescent carbohydrates, which can be used for determining glycosidase activity with a luminescent reporter molecule. The substrates were validated with glycosidase and revealed a clear linear range and enzyme-dependent signal upon the in situ generation of the luciferin moiety from the corresponding nitrile precursors. Besides, we showed that these compounds could directly be synthesized from unprotected glycosyl-α-fluorides in a two-step procedure with yields up to 75 %. The intermediate methyl imidate appeared a key intermediate which also reacted with d-cysteine to give the corresponding d-luciferin substrate rendering this a highly attractive method for synthesizing glycosyl luciferins in good yields.

Caged luciferins enable rapid multicomponent bioluminescence imaging.

Bioluminescence is a sensitive technique for imaging biological features over time. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable luciferase-luciferin pairs. Recent years have seen the development of numerous orthogonal probes for multi-parameter imaging. While successful, generating such tools often requires complex syntheses and lengthy enzyme evolution campaigns. This work showcases an alternative strategy for multiplexed bioluminescence that takes advantage of already-orthogonal caged luciferins and established uncaging enzymes. These probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm. Caged luciferins enabled two- and three-component imaging on the minutes time scale. We further showed that the tools can be used in conjunction with endogenous enzymes for multiplexed studies. Collectively, this approach lowers the barrier to multicomponent bioluminescence imaging and can be readily adopted by the broader community.

An emphatic study on the luciferin-luciferase bioluminescence system of Benthosema pterotum.

Approximately 80% of luminous organisms live in the oceans, and considerable diversity of life dependence on bioluminescence has been observed in marine organisms. Among vertebrates, luminous fish species are the only group of vertebrates that have the ability to emit bioluminescent light. Meanwhile, the lantern fish family (Myctophidae), with 33 genera all of which have the ability to emit light, is considered the most prominent family among the luminous fish of the deep oceans and seas. Lantern fish Benthosema pterotum has bioluminescence properties due to the presence of photophores scattered in its ventral-lateral region. However, no research has been performed on its bioluminescence system and light emission mechanism. The present research aimed to assess the type of bioluminescence, pigment, photoprotein, or luciferin-luciferase system in B. pterotum. In order to determine the type of light-emitting system in B. pterotum species, several specific experiments were designed and performed. It was shown that the light emission system in B. pterotum species is categorized into the luciferin-luciferase type. Conducting this research was not only innovative, but it also could be the beginning of further research in the field of marine biochemistry and production of the recombinant active forms of enzymes for industrial, commercial, medical, and pharmaceutical purposes.