Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay

A flexible, comparatively inexpensive system based on a liquid nitrogen-cooled slow-scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low-light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T₁ seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc -expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc -expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.     

Emerging tools for bioluminescence imaging

Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a widely used technique to interrogate gene expression and cell fate, both in small and large animal models of research. Recent developments include the generation of improved luciferase–luciferin systems for deeper and more sensitive imaging as well as new caged luciferins to report on enzymatic activity and other intracellular functions. Here, we critically evaluate the emerging tools for BL imaging aiming to provide the reader with an updated compendium of the latest developments (2018–2020) and their notable applications.     

Illuminating the Regenerative Properties of Stem Cells In Vivo with Bioluminescence Imaging

Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real-time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research.     

The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging.

In vivo bioluminescence imaging is becoming a very important tool for the study of a variety of cellular and molecular events or disease processes in living systems. In vivo bioluminescence imaging is based on the detection of light emitted from within an animal. The light is generated as a product of the luciferase-luciferin reaction taking place in a cell. In this study, we implanted mice with tumour cells expressing either a high or a low level of luciferase. In vivo bioluminescence imaging was used to follow tumour progression. Repeated luciferin injection and imaging of high and low luciferase-expressing tumours was performed. While low luciferase-expressing tumours grew similarly to vector controls, growth of the high luciferase-expressing tumours was severely inhibited. The observation that a high level of luciferase expression will inhibit tumour cell growth when an animal is subjected to serial in vivo bioluminescence imaging is potentially an important factor in designing these types of studies.     

In vivo bioluminescence imaging

In vivo bioluminescent imaging (BLI) is a versatile and sensitive tool that is based on detection of light emission from cells or tissues. Bioluminescence, the biochemical generation of light by a living organism, is a naturally occurring phenomenon. Luciferase enzymes, such as that from the North American firefly (Photinus pyralis), catalyze the oxidation of a substrate (luciferin), and photons of light are a product of the reaction. Optical imaging by bioluminescence allows a low-cost, noninvasive, and real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and treatment response. Bioluminescence in vivo imaging allows longitudinal monitoring of a disease course in the same animal, a desirable alternative to analyzing a number of animals at many time points during the course of the disease. We provide a brief introduction to BLI technology, specific examples of in vivo BLI studies investigating bacterial/viral pathogenesis and tumor growth in animal models, and highlight some future perspectives of BLI as a molecular imaging tool.     

Making the Brain Glow: In Vivo Bioluminescence Imaging to Study Neurodegeneration

Bioluminescence imaging (BLI) takes advantage of the light-emitting properties of luciferase enzymes, which produce light upon oxidizing a substrate (i.e., d-luciferin) in the presence of molecular oxygen and energy. Photons emitted from living tissues can be detected and quantified by a highly sensitive charge-coupled device camera, enabling the investigator to noninvasively analyze the dynamics of biomolecular reactions in a variety of living model organisms such as transgenic mice. BLI has been used extensively in cancer research, cell transplantation, and for monitoring of infectious diseases, but only recently experimental models have been designed to study processes and pathways in neurological disorders such as Alzheimer disease, Parkinson disease, or amyotrophic lateral sclerosis. In this review, we highlight recent applications of BLI in neuroscience, including transgene expression in the brain, longitudinal studies of neuroinflammatory responses to neurodegeneration and injury, and in vivo imaging studies of neurogenesis and mitochondrial toxicity. Finally, we highlight some new developments of BLI compounds and luciferase substrates with promising potential for in vivo studies of neurological dysfunctions.     

Chemically modified firefly luciferase is an efficient source of near-infrared light

Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of near-infrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.     

Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted micro-osmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted d-luciferin delivery enabled BLI for > or = 7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at < 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IkappaBalpha-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.     

活体动物体内光学成像技术的研究进展

生物发光和荧光成像作为近年来新兴的活体动物体内光学成像技术,以其操作简便及直观性成为研究小动物活体成像的一种理想方法,在生命科学研究中得以不断发展。利用这种成像技术,可以直接实时观察标记的基因及细胞在活体动物体内的活动及反应。利用光学标记的转基因动物模型可以研究疾病的发生发展过程,进行药物研究及筛选等。本文综述了现有活体动物体内光学成像技术的原理、应用领域及发展前景,比较了生物发光与几种荧光技术的不同特点和应用。     

A Causal Relation between Bioluminescence and Oxygen to Quantify the Cell Niche

Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner.     

Bacterial luciferase reporters: the Swiss army knife of molecular biology

Bioluminescence is a process during which light in the visible spectrum is emitted as a consequence of an enzymatic reaction catalyzed by luciferases. Luciferases have been identified mainly in marine organisms and are used for several biological purposes include camouflage, repulsion, attraction, communication and illumination. Some of the currently known luciferases have become indispensible tools in modern molecular biology and are used for diverse applications such as autoinducer-1 activity assays, promoter test assays in both prokaryotes and eukaryotes, imaging of bacterial infections in live animals, in vivo activity assays genes involved in host response and disease and monitoring of bacterial contaminations of food products. With the present review, the authors intend to give an overview on the currently used bacterial luciferase reporter systems, their methodologies and applications and compare them to other reporter systems.     

Cloning and mutagenetic modification of the firefly luciferase gene and its use for bioluminescence microscopy of engrailed expression during Drosophila metamorphosis

Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.     

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.     

The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.     

Factors influencing quantification of in vivo bioluminescence imaging: application to assessment of pancreatic islet transplants

The aim of this study is to determine and characterize factors influencing in vivo bioluminescence imaging (BLI) and apply them to the specific application of imaging transplanted pancreatic islets. Noninvasive quantitative assessment of transplanted pancreatic islets poses a formidable challenge. Murine pancreatic islets expressing firefly luciferase were transplanted under the renal capsule or into the portal vein of nonobese diabetic-severe combined immunodeficiency mice and the bioluminescence was quantified with a cooled charge coupled device camera and digital photon image analysis. The important, but often neglected, effects of wound healing, mouse positioning, and transplantation site on bioluminescence measurements were investigated by imaging a constant emission, isotropic light-emitting bead (lambda = 600) implanted at the renal or hepatic site. The renal beads emitted nearly four times more light than hepatic beads with a smaller spot size, indicating that light absorption and scatter are greatly influenced by the transplant site and must be accounted for in BLI measurements. Detected luminescence decreased with increasing angle between the mouse surface normal and optical axis. By defining imaging parameters such as postsurgical effects, animal positioning, and light attenuation as a function of transplant site, this study develops BLI as a useful imaging modality for quantitative assessment of islets post-transplantation.     

发光甲虫与生物荧光

生物荧光是活体生物自身可以发光的有趣生命现象。具有这一现象的生物存在于生物四界中,但目前关于这一现象的研究报道主要来自于昆虫,尤其是以萤火虫为代表的发光甲虫的研究。文章对发光甲虫的分类地位、生物荧光发生的原理、发光器官的类型、闪光的“开关”机制、生物荧光的生物学意义及其相关行为学研究进展等进行了详细介绍。此外,还简要提及了荧光生物及其荧光酶的应用。这对了解及探讨生物荧光现象、加强对中国的发光甲虫及其它发光生物的研究及保护利用具有一定的借鉴作用。     

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D-luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.     

Activity-based bioluminescence probes for in vivo sensing applications

Bioluminescence imaging is a highly sensitive technique commonly used for various in vivo applications. Recent efforts to expand the utility of this modality have led to the development of a suite of activity-based sensing (ABS) probes for bioluminescence imaging by ‘caging’ of luciferin and its structural analogs. The ability to selectively detect a given biomarker has presented researchers with many exciting opportunities to study both health and disease states in animal models. Here, we highlight recent (2021–2023) bioluminescence-based ABS probes with an emphasis on probe design and in vivo validation experiments.     

ANALYSIS OF NUCLEAR MUTANTS OF CHLAMYDOMONAS DEFICIENT IN THE ACCUMULATION OF SPECIFIC CHLOROPLAST RNAS

Bioluminescence is broadly distributed in marine dinoflagellates and has been intensively studied in Lingulodinium ( Gonyaulax ) polyedra. In this species, bioluminescence is regulated in a circadian fashion; the enzyme (luciferase) and the luciferin (substrate)-binding protein are synthesized and degraded on a daily basis. Synthesis of both proteins is regulated at the level of translation. The L. polyedra luciferase gene is composed of three contiguous domains that are greater than 75% identical at the nucleic acid level. Possible explanations for the high degree of sequence conservation include: (1) the domains evolved through a recent duplication event; (2) the sequence similarity is maintained by a molecular process such as gene conversion; or (3) there is a functional role associated with the primary nucleic acid sequence, such as in the translational regulation of luciferase expression. The phylogenetic relationship of dinoflagellates predicted from 18S rDNA genes provides a framework for examining the molecular evolution of the regulation of luciferase expression and of genes encoding luciferase and the luciferin-binding protein. In particular, we are examining the evolution of the circadian rhythm of bioluminescence and of luciferase abundance, the presence/absence of the luciferin-binding protein, and the molecular structure of the luciferase gene. We anticipate that this approach will distinguish between regions of the luciferase molecule that are conserved for enzyme function versus those concerned with the regulation of protein expression. In addition, it will provide insight into the evolution of the regulatory processes and pathways.     

Self-illuminating quantum dot conjugates for in vivo imaging

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.