A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples

A combination of a two-dimensional photon detector (double-microchannel plate) with single-photon sensitivity and an optical projection system that allows space-resolved quantitation of luminescent emissions from spatially extended objects is described. A "luminescent image" of the object focused onto the detector is accumulated over a preset time and stored in a digital frame memory from which photon counts over areas of interest can be read. In this study, the object consisted of a microtiter plate containing luminescent samples which was placed below a projecting lens (2.0/21 mm, 36 X 24-mm format camera lens) at a distance of 38.5 cm. Although geometry substantially limited photon collection, the sensitivity achieved was only 10X less than that obtained with a dedicated photon-counting luminometer. A slightly diminished photon collection from peripheral wells was apparently caused by the projection system and could be corrected arithmetically. Both chemically generated luminescence (ATP bioluminescence) and cell-derived, superoxide-dependent luminescence (with lucigenin as chemilumigenic probe) were detected with excellent spatial resolution and linearity of response over a wide range.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

Stable luciferase transfected cells for studying steroid receptor biological activity

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in Intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.     

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.     

Increasing lanthanide luminescence by use of the RETEL effect.

BACKGROUND: Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767-778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy. METHODS: The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac-labeled sample was allowed to dry. Both a conventional arc lamp and a time-gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image. RESULTS: With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes, as molecular diagnostics for cytological and histopathological microscopic imaging.     

Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.     

In Vitro Synthesis of Subunits of Bacterial Luciferase in an Escherichia coli System

RNA extracted from the luminescent bacterium Vibrio harveyi was translated in an Escherichia coli system. RNA from highly luminescent cells produced both α and β subunits of luciferase in vitro, as confirmed by immunoprecipitation and partial proteolysis. RNA from cells before induction of luminescence did not direct luciferase synthesis.     

Detection of bioluminescence from individual bacterial cells: a comparison of two different low-light imaging systems

Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.     

Low-light imaging technology in the life sciences

Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life science. These systems use advances in camera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in imageprocessing hardware and software have increased the usefulness of these systems in the biosciences. Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).     

Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors

Light emission from the bacterial luciferase operon has been variously exploited during last two decades. The use of convenient inducible promoters has granted significant degrees of specificity to whole cell-based assays for high-throughput screening and environmental monitoring. Nevertheless, unexplained unspecific responses have been repeatedly reported. Here, we show that the impairment of the intracellular biochemical equilibrium interferes with the luminescence produced by Escherichia coli and Staphylococcus aureus strains carrying the lux operon under constitutive or inducible control. Compounds as trimethoprim and methotrexate, by indirectly inducing NADPH accumulation, enhance light emission. Conversely, molecules driving the cell toward an oxidized state, as dimethyl sulfoxide, inhibit luminescence. These findings fit into the accepted biochemical pathway for bioluminescence, where NADPH and reducing equivalents are necessary for the production of luciferase substrates, although they do not directly take part into the light-emitting reaction. Moreover, we investigated the influence of induction timing upon the bioluminescence response from inducible reporter systems and demonstrated a correlation between the emitted light and the growth phase at which induction is performed. Our results provide explanations for some unspecific responses recorded so far in whole cell-based luminescent biosensors and emphasize the intrinsic limitations of this kind of reporting system.     

Bright stable luminescent yeast using bacterial luciferase as a sensor

Bright luminescent yeast cells with light intensities similar to bacteria containing luciferase (LuxAB) were generated by providing saturating nontoxic levels of the substrates for the bioluminescence reaction (FMNH(2)+O(2) and fatty aldehyde-->light). Z-9-Tetradecenal added to yeast (+luxAB) gave a luminescent signal close to that with decanal with the signal remaining strong for >24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min. Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal. Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the NADPH-specific FMN reductase (FRP) from Vibrio harveyi. The recognition of the nontoxicity and high luminescence generated by Z-9-tetradecenal and the generation of high levels of FMNH(2) in yeast by transformation with a flavin reductase provide evidence for the strong potential use of bacterial luciferase as the light-emitting sensor of choice in eukaryotic organisms.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. Resolution from luciferase and dependence on fatty acids

The enzyme responsible for the stimulation by ATP AND NADPH of light emission catalyzed by bacterial luciferase has been partially purified from extracts of the luminescent bacterium, Photobacterium phosphoreum. The stimulatory activity was found to be stabilized by high concentrations of mercaptoethanol, permitting it to be separated from luciferase into an active and stable form and enabling further characterization of its functional properties. The activity of the enzyme was shown to be dependent not only on ATP and NADPH but also on the presence of a long chain fatty acid, and was inhibited by the addition of NADH and horse liver alcohol dehydrogenase. The specificity for fatty acids, as measured by the stimulation of luciferase activity, had a very limited range, with maximal luminescence being obtained with myristic acid and lower responses being observed only with tridecanoic and pentadecanoic acid. These results provide evidence in vitro for an enzyme in bioluminescent bacteria that functions as a fatty acid reductase converting fatty acids to aldehydes which in turn can be utilized by luciferase in the light-emitting reaction.     

High Throughput Screening of Signal Transduction Mutants With Luciferase Imaging

A detailed procedure for high throughput genetic screening of hormone and environmental stress signal transduction mutants of Arabidopsis thaliana is described. The screen was carried out with mutagenized plants expressing the firefly luciferase reporter under control of a cold, osmotic stress, and absciscic acid responsive promoter. A thermoelectrically cooled CCD camera was used to detect luminescence emitted by the plants in response to stresses or ABA. Advantages of the screening procedure include high throughput, capability to identify low as well as high expression mutants and employment of a highly sensitive but affordable imaging system and software. This procedure can be used to study complex signal transduction networks in higher plants.     

Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6-12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.     

Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.     

Cloning and expression of genes of the luminescence system in Photobacterium leiognathi

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.