Chemiluminescence Emission Research Articles

Selective chemiluminescent TURN-ON quantitative bioassay and imaging of intracellular hydrogen peroxide in human living cells

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 μM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

Self-Enhanced Chemiluminescence of Tris(bipyridine) Ruthenium(II) Derivative Nanohybrids: Mechanism Insight and Application for Sensitive Silver Ions Detection.

In recent years, self-enhanced tris(bipyridine) ruthenium(II)-based luminescence systems have achieved great development in electrochemiluminescence (ECL) but are seldom mentioned in chemiluminescence (CL). Herein, a self-enhanced CL luminophore with excellent CL behavior was synthesized by covalently cross-linking tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) dichloride ([Ru(dcbpy)3]Cl2) with branched polyethylenimine (BPEI) in one molecule (BPEI-Ru(II)), which then self-assembled into nanoparticles (BRuNPs). The nanoparticles exhibited stable and strong CL emission with potassium persulfate (K2S2O8) as the oxidant. After the redox reaction between K2S2O8 and BRuNPs, and the subsequent intramolecular electron-transfer reaction, excited state luminophores were generated to emit light. This self-enhanced CL system shortened the electron transfer distance and reduced energy loss, thus improving the luminous efficiency. In addition, the CL lifetime of BRuNPs/K2S2O8 was longer than classical luminophores such as N-(4-aminobutyl)-N-ethylisoluminol (ABEI), indicating the potential application of this system in CL imaging. Surprisingly, Ag+ was found to greatly improve the CL efficiency of BRuNPs/K2S2O8 by catalyzing the decomposition of K2S2O8 to generate SO4•-. On the basis of the enhancement effect of Ag+, a simple and rapid CL method was proposed for Ag+ detection. The chemosensor showed a wide linear range from 25 to 3000 nM and low detection limit of 9.03 nM, as well as good stability and excellent selectivity. More importantly, this result indicated that Ag+ can be used as a coreaction accelerator to develop a ternary self-enhanced CL system, BRuNPs/K2S2O8/Ag+.

Immunoassays Using Optical-Fiber Sensor with All-Directional Chemiluminescent Collection.

While chemiluminescent optical-fiber sensors present essential advantages for immunoassays of biomarkers, an inherent limitation hampering their detection sensitivity is the low collection efficiency of chemiluminescent emission induced by samples, owing to the microspatial scale of an optical fiber for transmission of light via total internal reflection. Here we present a robust approach to overcome this limitation, based on a uniquely designed all-optical chemiluminescent collection vial (CC vial) by using a concave mirror and a coaxial tubular mirror as its bottom and wall, respectively. Using cardiac troponin I (cTnI), a highly specific but low abundance cardiac biomarker, as the test sample, we show that accurate assays can be achieved in a wide linear range of 1-80000 pg/mL. The limit of detection is as low as 0.31 pg/mL, which is about 2 orders of magnitude lower than that obtained by a normal chemiluminescent optical fiber sensor. The method is successfully applied for determination of cTnI in real human serum samples with good accuracy and repeatability. Our study shows that the method can perform an all-directional collection of the chemiluminescent emission, thus, greatly enhancing the collection efficiency and improving the sensitivity of the immunoassays. It is also worth mentioning that the proposed strategy requires neither complex equipment nor additional chemicals, making it a cost-effective and universal approach for ultrasensitive detection of trace biomarkers.

Unprecedented strong intrinsic chemiluminescence generation from degradation of halogenated hydroxy-quinoid pollutants by Co(II)-mediated advanced oxidation processes: The critical role of site-specific production of hydroxyl radicals

We found previously that an unusual two-step hydroxyl radical (OH)–dependent chemiluminescence (CL) can be produced by the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ, also known as p-chloranil) and H2O2. However, neither OH generation nor CL emission could be observed between H2O2 and chloranilic acid (CA), the dihydroxylation product of TCBQ. Here we found, unexpectedly, that distinct CL could be produced from CA/H2O2 in the presence of a variety of transition metal ions, among them Co(II) was found to be the most effective. Interestingly, the kinetic profiles of CL production, CA degradation, and reactive oxygen species (ROS) generation were found to be markedly changed with the molar ratios of Co(II):CA. When compared with the classic Fe(II)-mediated Fenton system, we found that the much stronger CL emission and more effective degradation of CA induced by Co(II)-mediated Fenton-like system is mainly due to the highly reactive OH generated site-specifically by Co(II)/H2O2, although other ROS such as O2− and 1O2 were also detected. More interestingly, not only CA, other halogenated anilic acids and catechols could also produce significant CL by Co(II)-mediated Fenton-like system. This is the first report on the distinct CL emission from degradation of halogenated hydroxyl-quinones by Co(II)-mediated Fenton-like advanced oxidation process, which is dependent on the site-specifically produced OH. These findings may have potential applications in measuring halogenated hydroxyl-quinones, and developing more effective techniques to degrade these types of important environmental pollutants.

Intensive and Persistent Chemiluminescence System Based on Nano-/Bioenzymes with Local Tandem Catalysis and Surface Diffusion.

A chemiluminescence (CL) system with long persistent and intensive emission is essential for accurate CL quantitative analysis and imaging assay. However, with most known CL systems being flash-type, it is still a great challenge to develop long-lasting CL systems. Here, by combining an iron porphyrin metal-organic frameworks (FePorMOFs) based peroxidase mimic with natural glucose oxidase (GOx), an intensive and persistent CL system is presented on the basis of local tandem catalysis and surface diffusion of the nano-/bioenzymes (FePorMOF/GOx). FePorMOF synthesized by iron porphyrin linker and zirconium ion node possesses high peroxidase catalytic activity and stability. Using luminol and glucose as substrate, the FePorMOF/GOx CL system can produce intensive CL emission containing a plateau period of 7.5 h. The strong CL signal is due to the local tandem generation and reaction of H2O2 by GOx and FePorMOF, which avoids the diffusion-limited kinetics and leads to a high catalytic efficiency of the nano-/bioenzymes. On the other hand, the long persistent CL emission is attributed mainly to the enzymatic reaction-controlled H2O2 supply and surface diffusion-controlled CL reaction. The proposed CL system is explored for CL imaging sensing of glucose and homogeneous immunoassay of α-fetoprotein. The nano-/bioenzymes CL system exhibits intensive and long constant CL emission in physiological condition, showing promising applications in real-time bioassay and bioimaging.

Efficient Cascade Resonance Energy Transfer in Dynamic Nanoassembly for Intensive and Long-Lasting Multicolor Chemiluminescence.

Light emission induced by chemical reactions, known as chemiluminescence (CL), has been widely used for bioassays, biosensors, imaging, and illumination applications. Most known CL systems exhibit flash-type single-color light emissions, which limit their applications. Long-lasting multicolor CL in aqueous solutions is highly desirable, especially for biological applications, but remains a challenge. Herein, we report a simple strategy of achieving highly efficient cascade Förster resonance energy transfer (FRET) in the dynamic nanoassembly of β-cyclodextrin (β-CD), CL reagents, and fluorophores in aqueous solution, which emits intensive multicolor CL with adjustable wavelength within 410-610 nm. β-CD can bind CL reagents and fluorophores to form a dynamic nanoassembly. These nanoassemblies can bring the included luminescent intermediate and fluorophores into close proximity and proper alignment, which should greatly enhance the FRET efficiency between luminescent intermediate and fluorophores. Indeed, the cascade FRET efficiency in this supramolecular nanoassembly reaches up to 92%, which is comparable with the cascade FRET systems based on covalently linked donors and acceptors. By using hydroxypropyl methylcellulose as the thickener to slow the diffusion (to elongate the CL emission), and using Ca(OH)2 solid (a low solubility strong base) as buffer to maintain the pH in the optimal range for the CL reaction, this nanoassembly system has been further developed to achieve slow-diffusion-controlled catalytic CL reactions, which enables long-lasting multicolor CL in aqueous solution that is visible to naked eyes and lasts for more than 20 h. The multicolor CL systems can be used to prepare transformable two-dimensional multicolor codes for encryption application.

In-Parallel Polar Monitoring of Chemiluminescence Emission Anisotropy at the Solid–Liquid Interface by an Optical Fiber Radial Array

Chemiluminescence (CL) detection is widely employed in biosensors and miniaturized analytical devices since it offers high detectability and flexible device design (there are no geometry requirements for the measurement cell, except the ability to collect the largest fraction of emitted photons). Although the emission anisotropy phenomenon for an emitting dipole bound to the interface between two media with different refractive index is well known for fluorescence, it is still poorly investigated for CL reactions, in which the excited-state reaction products can diffuse in solution before the photon emission event. In this paper, we propose a simple method for the real-time evaluation of the CL emission anisotropy based on a radial array of optical fibers, embedded in a poly(methyl methacrylate) semicylinder and coupled with a Charge-Coupled Device (CCD) camera through a suitable interface. The polar-time evolutions of the CL emission have been studied for catalyzing enzymes immobilized onto a solid surface (heterogeneous configuration) or free in solution (homogeneous configuration). Evidence of the anisotropy phenomenon is observed, indicating that the lifetime of the excited-state products of the enzyme-catalyzed reactions is shorter than the time required for their diffusion in solution at a distance at which the CL can be considered isotropic. These results open new perspectives in the development of CL-based miniaturized analytical devices.

Understanding chemiluminescence in catalytic oxidation of CO and hydrocarbons

A chemiluminescence (CL) instrument was developed to understand the CL behavior of catalytic oxidation of CO and hydrocarbons, which are major processes occurring in catalytic converters. The instrument is based on the cooperation among a gas mixer, a custom-made CL analyzer, and an on-line gas chromatography. The concept of the CL method was formulated by thoroughly investigating the CL emission during the catalytic oxidation of CO and C3H6 in the presence of O2 and/or NO under both stoichiometric and non-stoichiometric conditions. All the oxidation reactions were found to be CL-active, where the CL intensity increased exponentially with the temperature. The steady-state measurements showed good linearity between the CL intensity and the reaction rates, demonstrating that the CL emission is a direct product of the reactions. It was also found that the CL intensity per CO2 production was different among the reactions, most plausibly due to both/either the presence of multiple CL processes and/or the difference in the heat of the reactions. Based on the linear correlation between the CL intensity and the reaction rate, a feasibility study was successfully demonstrated for rapid catalyst evaluation in C3H6 oxidation.

A chemiluminescence reaction consisting of manganese(IV), sodium sulfite, and sulfur- and nitrogen-doped carbon quantum dots, and its application for the determination of oxytetracycline.

Sulfur- and nitrogen-doped carbon quantum dots (S,N-CQDs) were prepared by a solid-phase hydrothermal method from cysteine and citric acid and characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, and FTIR spectroscopy. These QDs were exploited as enhancers to amplify the chemiluminescence (CL) of manganese(IV)-sodium sulfite reaction. S,N-CQDs exceptionally enhanced the CL intensity of this system, around 900-fold. This effect was attributed to the energy transfer from SO2*, produced by reaction of Mn(IV) with SO32-, to S,N-CQDs. The maximum wavelength of CL emission was 480nm, which confirmed that the final emitting species was S,N-CQDs. After optimization of reaction conditions, the analytical applicability of S,N-CQD-Mn(IV)-SO32- CL system was studied. In the presence of oxytetracycline, the CL intensity was significantly diminished. A linear relationship was observed between CL signal and the logarithm of oxytetracycline concentration in the range of 0.075-3.0μM with a detection limit of 25nM. This CL assay for oxytetracycline was used for analysis of spiked milk and water samples. Graphical abstractSchematic representation of the amplified chemiluminescence (CL) reaction consisting of sulfur- and nitrogen-doped carbon quantum dots (S,N-CQDs) Mn(IV) and Na2SO3. Sub-micromolar levels of oxytetracycline can be determined by using this system.

Chemiluminescence as a diagnostic in studying combustion instability in a practical combustor

Prediction of combustion instability is essential in reducing development costs of large scale rocket engines. Recently, high fidelity CFD models have shown some ability to represent the stability characteristics of sub-scale experimental combustors. A challenge remains in acquiring experimental measurements of critical combusting flow properties, especially measurements of the unsteady heat addition field, for comparison to models. The chemiluminescent emission of radicals OH* and CH*, although commonly employed, may fall short due to its known dependence on pressure, strain rate, equivalence ratio, and turbulence level. Since these flame properties have large variations in an unstable rocket combustor, it is difficult to decouple the emission from all other variables except the heat release. Computations capture the local variations, and therefore can be used for a direct comparison with the measurements. The present study investigates the relationship between chemiluminescent species and heat release rate of the flame using simulations that employ detailed kinetics including the emitting species. This relationship is then compared to experimental spectral measurements captured with a fiber optic probe to check the validity of the model and to provide insight on the ability of the chemiluminescent light emission to indicate heat release in a high pressure unstable flame.

Correlation analysis of chemiluminescent and pollutant emissions of a liquid-fueled turbulent swirl burner

Real-time combustion and emission control is an ongoing challenge in combustion technology and science. Hence, the scope of the present paper is the investigation of the relationship between the chemiluminescent signal and the CO and NOX emissions. Flame emission spectrometry measurements were carried out to determine the characteristic free radicals of the spectra. For the experiments, a lean premixed liquid fuel burner equipped with an airblast atomizer was used in a test rig at 15 kW combustion power. The following measurement parameters were modified: combustion air flow rate, atomizing pressure, and the vertical alignment of the spectrometer. Furthermore, various half-cone angle quarls were mounted on the burner lip to extend the lean flame blowout stability limit. The CO and NOX emissions and the chemiluminescence intensity ratios of the strongest peaks of OH*, CH*, C2*, HCO*, and CH2O* were evaluated separately at first. Then a correlation analysis of the intensity ratios and the pollutant emission components was carried out. A notable linear correlation was found between both the HCO*/C2* and OH*/C2* intensity ratios and the CO emission in certain parameter combinations.

Ultrasensitive haptoglobin biomarker detection based on amplified chemiluminescence of magnetite nanoparticles

BackgroundHaptoglobin is an acute-phase protein used as predicting diagnostic biomarker both in humans (i.e., diabetes, ovarian cancer, some neurological and cardiovascular disorders) and in animals (e.g., bovine mastitis). The latter is a frequent disease of dairy industry with staggering economical losses upon decreased milk production and increased health care costs. Early stage diagnosis of the associated diseases or inflammation onset is almost impossible by conventional analytical manners.ResultsThe present study demonstrates a simple, rapid, and cost-effective label-free chemiluminescence bioassay based on magnetite nanoparticles (MNPs) for sensitive detection of haptoglobin by employing the specific interaction of hemoglobin-modified MNPs. The resulting haptoglobin-hemoglobin complex inhibits the peroxidase-like activity of luminol/H2O2-hemoglobin-MNPs sensing scheme and reduces the chemiluminescence intensities correspondingly to the innate haptoglobin concentrations. Quantitative detection of bovine haptoglobin was obtained within the range of 1 pg mL−1 to 1 µg mL−1, while presenting 0.89 pg mL−1 limit of detection. Moreover, the influence of causative pathogenic bacteria (i.e., Streptococcus dysgalactiae and Escherichia coli) and somatic cell counts (depicting healthy, sub-clinical and clinical mastitis) on the emitted chemiluminescence radiation were established. The presented bioassay quantitative performances correspond with a standardized assay kit in differentiating dissimilar milk qualities.ConclusionsOverall, the main advantage of the presented sensing concept is the ability to detect haptoglobin, at clinically relevant concentrations within real milk samples for early bio-diagnostic detection of mastitis and hence adjusting the precise treatment, potentially initiating a positive influence on animals’ individual health and hence on dairy farms economy.

Nanomaterial-enhanced chemiluminescence reactions and their applications.

Chemiluminescence (CL) analysis is a trace analytical method that possesses advantages including high sensitivity, wide linear range, easy operation, and simple instruments. With the development of nanotechnology, many nanomaterial (NM)-enhanced CL systems have been established in recent years and applied for the CL detection of metal ions, anions, small molecules, tumor markers, sequence-specific DNA, and RNA. This review summarizes the research progress of the nanomaterial-enhanced CL systems the past five years. These CL reactions include luminol, peroxyoxalate, lucigenin, ultraweak CL reactions, and so on. The CL mechanisms of the nanomaterial-enhanced CL systems are discussed in the first section. Nanomaterials take part in the CL reactions as the catalyst, CL emitter, energy acceptor, and reductant. Their applications are summarized in the second section. Finally, the challenges and opportunities are discussed.

On the chemiluminescence emission of luminol: protic and aprotic solvents and encapsulation to improve the properties in aqueous solution.

Luminol is a popular molecule that is currently gaining further interest due to its potential role for non-invasive cancer treatments. Design of more efficient derivatives in this context would benefit from a clear knowledge on the origin of the distinct intensity and spectroscopic properties in protic and aprotic solvents observed experimentally, which are still not rationalized. By efficiently combining molecular dynamics, quantum methodologies based on density functional theory and multiconfigurational quantum chemistry and hybrid approaches, and developing herein a computational approach for accurately determining "molar negative extinction (or gain) coefficients of emission", we firstly demonstrate that the amino and imino forms of the 3-aminophthalate dianion are responsible for the chemiluminescence in protic and aprotic medium, respectively. Secondly, we show that the coupling between the adjacent amino and carboxylate groups of luminol existing in aprotic solvents must be kept in aqueous solution to increase the chemiexcitation and emission intensity. Thirdly, modifications of luminol are proposed and simulated showing improved performances as compared to the parent molecule (stronger emission electronic transition and longer emission wavelengths) under the physiological conditions of relevance in biological and medical applications.

Black phosphorus quantum dots as novel electrogenerated chemiluminescence emitters for the detection of Cu2.

For the first time, electrochemiluminescence (ECL) emission was observed from black phosphorus quantum dots (BPQDs) in the presence of K2S2O8 as the co-reactant. The potential application of BPQDs ECL in analytical chemistry was also demonstrated using Cu2+ as an example.

Synergistic chemiluminescence nanoprobe: Au clusters-Cu2+-induced chemiexcitation of cyclic peroxides and resonance energy transfer.

An interesting chemiluminescence (CL) phenomenon of cyclic peroxides originating from tetrahydrofuran hydrogen peroxide (THF-HPO) in the presence of BSA-stabilized Au NCs (Au@BSA NCs) was found for the first time. In this CL system, Au@BSA NCs can greatly accelerate the decomposition of THF-HPO, and then chemiluminescence resonance energy transfer (CRET) occurs between excited dioxetane derivatives and the Au@BSA NCs, yielding enhanced CL emission which can be further enhanced more than 10 times by the addition of copper ions. Based on this, a synergistic CL nanoprobe with a special signal amplification strategy was developed.

Effects on performance, combustion and pollutants of water emulsified fuel in an aeroengine combustor

The present work provides an experimental investigation of the use of water emulsified fuels to control the combustion performance and reduce nitrogen oxides emissions into a Jet-A1 fueled gas turbine combustor. Experiments have been carried out using a test rig equipped with a 300-kW liquid-fueled swirling burner. Several fuel-to-air ratios have been tested in combination with various water concentrations. Measurements of exhaust emissions have been performed. Furthermore, high-speed cameras in visible and ultraviolet spectral ranges, have been used. The snapshot Proper Orthogonal Decomposition of the flame images of both broadband emission and hydroxyl radical chemiluminescence has allowed to detect the most relevant flame structures, in combination with the modal frequency spectra.Results figured out that, the addition of water in the fuels led to lower combustion temperature and consequently to lower thermal nitrogen oxides than the case of neat fuel. On the other hand, the thermal efficiency significantly dropped in presence of high-water content (5% H2O) and ultra-lean conditions, while it remained acceptable at 2.5% H2O and fuel rich conditions. Furthermore, under the near-lean blowout condition, the flame becomes very unstable and flame oscillations take place in the axial direction. This combines with the increase in the relative energy of the first Proper Orthogonal Decomposition modes. Finally, the phase space analysis of modes 1–2 of the hydroxyl radical chemiluminescence emission defined a criterion for the detection of the establishment of the flame instability, which corresponds to phase angles ranging between -π/6 and π/6.

Black phosphorus quantum dots sensitized lucigenin chemiluminescence in mild alkaline condition and its application in sensitive detection of Co2+

As a novel 2-dimensional nanomaterials, black phosphorus quantum dots (BPQDs) have attracted many interests in recent years. In the present work, the potential application of BPQDs in lucigenin chemiluminescence (CL) was researched. Under mild alkaline condition, lucigenin CL was extremely weak, and was difficult for analytical application. Both BPQDs and Co2+ exhibited enhancing effect on lucigenin CL due to their catalytic effects. The synergic effect of BPQDs and Co2+ could generate the strongest CL emission in weak alkaline solution, which provided the possibility for higher sensitive detection of Co2+. Several impacting factors, such as the concentrations of lucigenin and H2O2, the pH value, the mixing sequence of reagents, and the diluted ratio of BPQDs were investigated. Under the optimal condition, Co2+ was sensitively detected in the range of 8.0 × 10−9 to 8.0 × 10−8 mol L − 1, and the detection limit was determined as 3.0 × 10−9 mol L − 1 (3σ). The proposed method exhibited higher sensitivity, wider linear range, and good selectivity compared with traditional CL method. Finally, the mechanism for BPQDs sensitized lucigenin CL was discussed.

Luminescent metal organic frameworks–based chemiluminescence resonance energy transfer platform for turn–on detection of fluoride ion

A luminescent metal organic frameworks (MOFs)–based chemiluminescence resonance energy transfer (CRET) platform was constructed for turn–on detection of fluoride ion. A hybrid MOFs was prepared by encapsulating strong fluorescence 2′,7′–dichlorofluorescein (DCF) into the frames of NH2–MIL–101(Al) MOFs, which led to a significant suppression of fluorescence signal of DCF. In the presence of fluoride ion, it destroyed the structure of the hybrid MOFs and released DCF molecules from the frames due to the formation of more stable aluminum hexafluoride complex ions [AlF63−] between fluoride ion and aluminum ion. The released DCF molecules accepted the energy originating from the chemical reaction of bis(2,4,6–trichlorophenyl)oxalate (TCPO) with hydrogen peroxide (H2O2), producing a strong chemiluminescence (CL) emission. The CL signal was strong dependent on the concentration of fluoride ion presented and showed a linear response in the range of 0.5–80.0 μmol L−1 (9.5 μg L−1–1.52 mg L−1). The detection limit was 0.05 μmol L−1 (about 0.95 μg L−1) fluoride ion and the relative standard deviations was 2.3% for 40.0 μmol L−1 fluoride ion solution (n = 11). This MOFs–based CRET method was successfully applied to the determination of fluoride ion in drinking water samples, demonstrating its potential application in analysis of real samples.

Two-line ([formula omitted]/[formula omitted]) chemiluminescence technique for equivalence ratio mapping in turbulent stratified flames

Equivalence ratio fluctuations play an important role in exploring and controlling flame dynamic instabilities such as thermoacoustic combustion instabilities associated with lean premixed gas turbine (GT) engines. However, it is difficult to reliably measure local equivalence ratio, and so we often make estimates based on the ratios of chemiluminescent emissions. Recent studies have reported that equivalence ratio varies with chemiluminescence intensity ratio of CH* and CO2* in the visible spectrum (431 and 410 nm, respectively). A methodology based on this relationship is adopted in the present study to construct equivalence ratio maps for a series of turbulent premixed and stratified methane/air flames under globally lean conditions (mean or global equivalence ratio, ϕ¯=0.75), over a range of stratification with ϕ spanning 0.375–1.125 for the highest level of stratification. In contrast with the previously used CH*/OH* method, the current technique allows for a single visible range camera to be used for chemiluminescence imaging. A comparison of the current estimated equivalence ratio with previously calculated local equivalence ratio from Raman-scattering measurements shows that the two-line chemiluminescence technique can be reliably used to determine equivalence ratio in the instantaneous reaction zone of turbulent flames even at higher turbulence levels and mixture stratification conditions.