The light of the firefly under the influence of ethyl acetate

When a firefly is made to inhale ethyl acetate vapour, a constant glow appears after a few minutes from its abdominal lantern. This control experiment has been performed by a few workers to record the emission spectrum of the firefly. However, a time-resolved experiment performed by us on this continuous light emitted by the species Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae) reveals that it is composed of a continuous train of tiny pulses! The nature of the pulses suggests that an oscillatory chemical reaction continues in the microsecond time scale in the lantern of the anaesthetized firefly.     

Sharp intense line in the bioluminescence emission of the firefly

Numerous investigations have been carried out on the spectral distribution of the light of different species of fireflies. Here we record the emission spectrum of the Indian species of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) on a color film. Green and red color-sectors, with an intense yellow one in between, appear in this spectrum. Intensity profile of this spectrum reveals a hitherto undetected strong narrow yellow line, which lies within the full-width-at-half maximum (FWHM) of the intensity profile. The spectrum recorded in a high-resolution spectrometer confirms the presence of this sharp intense line. This finding lends support to an earlier drawn analogy between the in vivo emission of the firefly and laser light.     

Monitoring of spatial expression of firefly luciferase in transformed zebrafish

The firefly luciferase gene attached to the cytomegalovirus promoter was transferred into zebrafish (Brachydanio rerio) by microinjection of fertilized eggs. Light emission could be monitored in vivo in eggs and throughout the early development of the fish by low-light video-image analysis. Gene expression was transient in most of the cases lasting for about 2 weeks. This gene cassette proved to be a very convenient and nondestructive transformation marker and the firefly luciferase gene appears to be a powerful tool for real-time imaging of tissue-specific gene expression in transgenic fish.     

Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase‐encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch‐culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram‐negative Escherichia coli::luxAB strains and a Gram‐positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491–500 nm (± 5 nm) and a second peak at 585–595 (± 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550–650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram‐positive and Gram‐negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright © 2003 John Wiley & Sons, Ltd.     

The chromophore of pholasin: A highly luminescent protein

Pholasin is the photoprotein extracted from the marine bivalve Pholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin. Pholasin could not be reactivated using chromophores from the hydroid Obelia geniculata (coelenterazine) and from the ostracod shrimp Vargula (formerly Cypridina) hilgendorfi. Furthermore, the reaction product of the Vargula chromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (γ_max 480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of < 2000. Fluorescence masurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence.     

Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.     

FLUORESCENCE EMISSION SPECTRA OF MARINE AND BRACKISH‐WATER ECOTYPES OF FUCUS VESICULOSUS AND FUCUS RADICANS (PHAEOPHYCEAE) REVEAL DIFFERENCES IN LIGHT‐HARVESTING APPARATUS1

The Bothnian Sea in the northerly part of the Baltic Sea is a geologically recent brackish‐water environment, and rapid speciation is occurring in the algal community of the Bothnian Sea. We measured low‐temperature fluorescence emission spectra from the Bothnian Sea and the Norwegian Sea ecotypes of Fucus vesiculosus L., a marine macroalga widespread in the Bothnian Sea. Powdered, frozen thallus was used to obtain undistorted emission spectra. The spectra were compared with spectra measured from the newly identified species Fucus radicans Bergström et L. Kautsky, which is a close relative of F. vesiculosus and endemic to the Bothnian Sea. The spectrum of variable fluorescence was used to identify fluorescence peaks originating in PSI and PSII in this chl c–containing alga. The spectra revealed much higher PSII emission, compared to PSI emission, in the Bothnian Sea ecotype of F. vesiculosus than in F. radicans or in the Norwegian Sea ecotype of F. vesiculosus. The results suggest that more light‐harvesting chl a/c proteins serve PSII in the Bothnian Sea ecotype of F. vesiculosus than in the two other algal strains. Treatment of the Bothnian Sea ecotype of F. vesiculosus in high salinity (10, 20, and 35 practical salinity units) for 1 week did not lead to spectral changes, indicating that the measured features of the Bothnian Sea F. vesiculosus are stable and not simply a direct result of exposure to low salinity.     

Thein vivo pattern of firefly luciferase expression in transgenic plants

Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether inin vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and thein vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, thein vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.     

Bioluminescence in firefly larvae: A test of the aposematic display hypothesis (Coleoptera: Lampyridae)

We investigated the hypothesis that bioluminescence in firefly larvae (Coleoptera: Lampyridae) functions as an aposematic display. In two experiments, we confirmed the distastefulness of firefly larvae, and tested the hypothesis that a naive, nocturnal predator can learn to use light signals as aposematic cues for avoiding distasteful prey. Larvae were rejected as acceptable prey by 100% of the house mice (Mus musculus) tested. Mice learned to avoid bitter food associated with light cues significantly faster (P=0.003) than mice presented with food lacking light cues. We conclude that luminescent glowing in firefly larvae meets the requirements of an aposematic signal.     

Isolation and characterization of luciferase from Indian firefly,Luciola praeusta

Luciferase from Indian firefly Luciola praeusta (Coleoptera: Lampyridae: Luciolinae) was isolated and the properties compared with that of the North American firefly, Photinus pyralis. Luciola praeusta luciferase was purified using acetone extraction, gel‐filtration column chromatography, ammonium sulfate precipitation and anion exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicates a homogeneous preparation and the molecular mass was slightly higher than that of Photinus pyralis. The effect of pH, buffer composition and metal ions on the spectral characteristics was studied. The maximum bioluminescence activity of luciferase was observed in ACES buffer at pH 6.5. The emission maximum of 562 nm (in crude extract) was red shifted to 570 nm in Tricine buffer at pH 7.8. In addition, the effect of bovine serum albumin on the storage stability of the protein was investigated. Based on the unique spectral characteristics observed, we propose that Luciola praeusta luciferase in the native form is suitable for the assay of biochemical metabolites in acidic pH. Copyright © 2013 John Wiley & Sons, Ltd.     

Extended‐release PEG–luciferin allows for long‐term imaging of firefly luciferase activity in vivo

Bioluminescence has gained favour in the last decade as an approach for observing tumours in vivo in a non‐destructive manner. This very sensitive technique is based on light emission by the reaction of luciferin with the enzyme luciferase, as measured by a photodetector. Ever since the development of recombinant tumour cell lines that have been engineered to produce luciferase, a vast number of experiments have been carried out examining tumour growth, tumour metastasis and the effect of therapeutic regimens in such cases. A primary stumbling block, however, is the relatively short circulatory half‐life of luciferin. In this paper, we propose the PEGylation of 6‐amino‐d ‐luciferin to extend its in vivo circulatory half‐life, thus making the possibility of long‐term observations in animals possible. The covalent attachment was through a carbamate linker that is known to hydrolyse in vivo, releasing the parent compound. Based on our studies, longer emission of the PEGylated luciferin was observed, as compared to free luciferin in mice bearing PC3 prostate tumours expressing luciferase. This result suggests that this reagent can be used in applications requiring extended monitoring of luciferase activation in vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Cu‐doped quantum dots: a new class of near‐infrared emitting fluorophores for bioanalysis and bioimaging

Transition metal ion‐doped quantum dots (QDs) exhibit unique optical and photophysical properties that offer significant advantages over undoped QDs, such as larger Stokes shift to avoid self‐absorption/energy transfer, longer excited‐state lifetimes, wider spectral window, and improved chemical and thermal stability. Among the doped QDs emitters, Cu is widely introduced into the doped QDs as novel, efficient, stable, and tunable optical materials that span a wide spectrum from blue to near‐infrared (NIR) light. Their unique physical and chemical characteristics enable the use of Cu‐doped QDs as NIR labels for bioanalysis and bioimaging. In this review, we discuss doping mechanisms and optical properties of Cu‐doped QDs that are capable of NIR emission. Applications of Cu‐doped QDs in in vitro biosensing and in in vivo bioimaging are highlighted. Moreover, a prospect of the future of Cu‐doped QDs for bioanalysis and bioimaging are also summarized.     

Luminescence studies on Ba2ZnSi2O7 : Eu2+ phosphors crystals

A novel blue green‐emitting phosphor Ba₂ZnSi₂O₇ : Eu²⁺ was prepared by combustion synthesis method and an efficient bluish green emission under from ultraviolet to visible light was observed. The emission spectrum shows a single intensive band centered at 503 nm, which corresponds to the 4f⁶5d¹ → 4f⁷ transition of Eu²⁺. The excitation spectrum is a broad band extending from 260 to 465 nm, which matches the emission of ultraviolet light‐emitting diodes (UV‐LEDs). The effect of doped Eu²⁺ concentration on the emission intensity of Ba₂ZnSi₂O₇ : Eu²⁺ was also investigated. The result indicates that Ba₂ZnSi₂O₇ : Eu²⁺ can be potentially useful as a UV radiation‐converting phosphor for white light‐emitting diodes. Copyright © 2009 John Wiley & Sons, Ltd.     

Spectral properties of a long-wavelength absorbing form of protochlorophyll in seeds of Cyclanthera explodens

The in vivo absorbance spectrum of the inner seed coat of Cyclanthera explodens Naud. showed a main peak in the red region at 671 nm and a weak shoulder at about 640 nm. The pigments were extracted with acetone. separated by paper chromatography and analysed spectrophotometrically. The only detectable pigment was protochlorophyll. The in vivo fluorsecence emission spectra had two main peaks, one at 632 and one at 691 nm. The relation between the two peaks was changed when the exvcitation wavelength was altered from 440 to 460 nm. Excitation at 420 nm gave an additional fluorescence emission peak at 595 nm. These data indicate the presence of at least three forms of protochlorophyll in the Cyclantera seed coat. The spectrum of circular dichroism had a very intence and characteristic signal in the red region with a negative asymmetrical Cotton effect (664 (+), 669 (0) and 687 (?) nm). This indicates that at least one of the protochlorophyll forms is present in a more or less crystalline form.     

Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed

The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.     

An Optimized Triple Modality Reporter for Quantitative In Vivo Tumor Imaging and Therapy Evaluation

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.     

Synthesis and characterization of erbium‐doped YAlO3 phosphor

In the yttrium aluminium system, the YAlO₃ phosphor is a prominent host because of the yttrium aluminium ratio (1:1). Phosphor was synthesized by the solid‐state reaction method at variable concentrations of erbium (0.1–2.5 mol%). This method is suitable for large‐scale production and is a less time‐consuming method when compared with the soft synthesis method. The prepared sample was characterized by X‐ray diffraction technique and the crystallite size was calculated by Scherer's formula. Vibrational and bending analysis of prepared phosphor for optimized concentration of erbium ion is described based on the Fourier transform infrared spectroscopic technique. The photoluminescence (PL) emission spectra of prepared phosphor for variable concentrations of erbium ion were recorded and the excitation spectrum was found to be at 291 nm with three shoulder peaks at 305, 270 and 242 nm. For 291 nm excitation, the emission spectrum was found at 546 nm and 552 nm. PL intensity increased with increasing concentrations of erbium and after 2 mol% emission intensity decreased due to concentration quenching. Spectrophotometric determination of YAlO₃:Er³⁺ is described by CIE co‐ordinates and shows an intense emission in the green region such that the prepared phosphor can act as a single host for green light emission. Thermoluminescence glow curve analysis of the YAlO₃:Er³⁺ phosphor was recorded for different ultraviolet (UV) light exposures and gamma exposure. Different gamma doses 0.5–2 kGy show a linear response. Kinetic parameters were calculated by the peak shape method. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours

Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue‐shifted examples in each family, confirming the trend that the most blue‐shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases).     

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

The diversity in substrate recognition spectra exhibited by various β‐lactamases can result from one or a few mutations in the active‐site area. Using Escherichia coli TEM‐1 β‐lactamase as a template that efficiently hydrolyses penicillins, we performed site‐saturation mutagenesis simultaneously on two opposite faces of the active‐site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended‐spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K_M) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k_cat) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM‐1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.     

<Emphasis Type="Italic">In vivo</Emphasis> molecular imaging of experimental joint inflammation by combined <Superscript>18</Superscript>F-FDG positron emission tomography and computed tomography

Introduction  

The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation.