why is fluorescence quenching important

Then the slurry is removed by centrifugation and the clear liquid is evaluated by measuring the optical density at 360 nm or at 385 nm for DNP proline. For example, one can find out whether the interaction between the two proteins is cooperative or not, etc… Two examples to illustrate this, we used 2,p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescent probe to study the interaction between cytochrome c and cytochrome b2 core and to follow the binding of cytochrome b2 core on flavodehydrogenase. Greenwood, Michael. A hydrophobic interaction between the guest molecules and the dansyl unit of the sensors is suggested. By continuing to browse this site you agree to our use of cookies. 2 and Table 1. Michael graduated from Manchester Metropolitan University with a B.Sc. Greenwood, Michael. Fluorescence 2 of 4 quenching include concentration quenching (a decrease in the fluorescence-per-unit-concentration as the concentration is increased), also referred to as an inner filter effect, and chemical quenching. with these terms and conditions. Fig. Since oxygen diffusion in proteins is anisotropic and does not take one precise path, entry and exit rate constants should be considered as mean values of the different paths that oxygen molecules take to enter and to exit from the protein. There are two different ways of quenching: static and dynamic quenching.It is to be mentioned that there is a difference between quenching and decrease of fluorescence due to the high state of molecular excitement or chemical changes of FLUOROPHORE (like oxidation). The fluorescence quenching of Pe and derivatives has been investigated by fluorescence upconversion. By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. Fluorescence quenching has been widely studied both as a fundamental phenomenon, and as a source of information about biochemical systems. He is currently completing a Ph.D. on the design and production of gold nanoparticles able to act as multimodal anticancer agents, being both drug delivery platforms and radiation dose enhancers. The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical. The students are able to determine that the fluorescence is quenched dynamically by collision with the halide, not by forming an equilibrated complex. Luckily, it can be understood at a basic level that’s relatively straightforward. For most samples, measurements were carried out at three different wavelengths (438, 475, and 490 nm). Quenching also results in reduced fluorescence intensity and frequently is brought about as a result of oxidizing agents or the presence of salts of heavy metals or halogen compounds. Going from the fact that the total fluorophore concentrations is, and that bound fluorophore does not fluoresce, replacing Equation 4.18 in 4.17 yields, Since the concentrations are proportional to the fluorescence intensities, Eq.4.19 can be written as, If we consider the concentration of bound quencher very small compared to the added quencher concentration, then free quencher concentration is almost equal to the total added concentration. We use cookies to help provide and enhance our service and tailor content and ads. An excited dimer is made up of two molecules with excited electrons or excimer (excited monomer). The quantum efficiency describes the efficiency of the process and is defined as the ratio of absorbed and emitted photons [13]. Also, binding parameters of the fluorophore-macromolecule complex can be determined by following fluorescence modification of the fluorophore observables. 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Selecting fluorescent probes that have fluorescence emission well separated spectrally from the absorption spectra of the quencher can circumvent this problem. Fluorescence quenching can also take place by the formation at the ground state of a non-fluorescent complex. News-Medical. Physikalisch-chemisches Praktikum I Fluorescence Quenching { 2016 is a very important step in many photocatalytic reactions, for example for solar fuel production. This can occur over distances of around 10 nm but becomes more likely the closer the dipoles are to one another at the rate of the inverse of the distance to the power of six. Stokes also discovered the wavelength shift to longer values in emission spectra that bears his name. Figure 36. 1984) showed that at low oxygen concentration (τo = 18.45 ns), kq is equal to 0.23 × 109 M− 1 s− 1 while at higher oxygen concentration (τo is taken as 15.375 ns calculated at 116.7 mM of oxygen), kq is equal to 1.12 × 109 M− 1 s− 1. The variation of the Trp residues intensity and lifetime are analyzed by plotting the intensities and the lifetimes in the absence and presence of TNS as a function of TNS concentration (Fig. We’ll have a look at these and more questions in this article. Quenching. Typical fluorescence decays and best multiexponential fits are shown in Fig. Quenching of Fluorescence with Temperature TN_P27; 15 Feb 2016, Georgios Arnaoutakis, Dirk Nather Reliable fluorescence standards and stable fluorescent probes for bio-analytics and chemistry are as important as sensitive indicator fluorophores that utilise the outstanding property of fluorescence of being highly sensitive to the Thus, the studied domain is much more restricted than in the case of longer fluorescence lifetimes. Static quenching is caused by the formation of a complex between a fluorescent and a quenching molecule. Quenching also results in reduced fluorescence intensity and frequently is brought about as a result of oxidizing agents or the presence of salts of heavy metals or halogen compounds. Pharm., Ph.D, ... Dr.Anup Kumar Das M. This allows the excited electron of the donor molecule to move to an unoccupied orbital of the acceptor molecule, becoming its ground state. High quantum yield (> 30%) and fast response (< 100 ps) detectors are needed to detect the weak fluorescence and resolve the short photon delay time in the photon-by-photon approach.96 To use an externally introduced fluorescent probe to measure angstrom-scale distance changes, the attachment of the probe is critical. We can notice that at low oxygen concentration kq value is 68% lower than that observed in the same, conditions in the absence of sucrose. Peng Chen, Nesha May Andoy, in Comprehensive Natural Products III, 2010. The fluorophore should be bound to one of the two proteins only (case of the extrinsic fluorophores) or should be part of it (case of the intrinsic fluorophores) if we want to perform such experiments. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/B9780444514493500046, URL: https://www.sciencedirect.com/science/article/pii/B978012409547212623X, URL: https://www.sciencedirect.com/science/article/pii/B9780081026908001810, URL: https://www.sciencedirect.com/science/article/pii/B9780444516565500682, URL: https://www.sciencedirect.com/science/article/pii/B9780444514493500095, URL: https://www.sciencedirect.com/science/article/pii/B9780128023259000094, URL: https://www.sciencedirect.com/science/article/pii/S1574064107800118, URL: https://www.sciencedirect.com/science/article/pii/B9780124095472109850, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2016, Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies. Fluorescence Spectroscopy Fluorescence quenching is an important technique for measuring binding affinity between ligands and proteins. Dr.Subhash C. Mandal M. Quenching of fluorescence is a physicochemical PROCESS that decreases fluorescent intensity of light emitting molecules. Studies performed in presence of 40% sucrose (Jameson et al. (accessed January 07, 2021). Excitation was performed with the frequency-doubled output of a Ti:Sapphire amplifier. Fluorescence lifetime of the intrinsic fluorophore is not modified. The quenching trend is separate from the objective of this experiment, though it heavily affects the process of fluorescence determination in general. Kedde reagent: Immediately on spraying, cardenolides form a pink or blue–violet color. The complex, once formed, is non-fluorescent. The dynamic quenching we described in the previous three sections occurs within the fluorescence lifetime of the fluorophore, i.e. This type of complex is called static quenching and it can be described with the following equations: where [FQ] is the complex concentration, and [F]1 and [Q]1 the concentrations of free fluorophore and quencher. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence (such as NADH, tryptophan or endogenous chlorophyll, phycoerythrin or green fluorescent protein). Thus, the kinetic constants measured at high oxygen concentrations (low fluorescence lifetimes) reflect also the diffusion of oxygen through the protein matrix, within an area smaller than that observed at low oxygen concentrations (higher lifetimes). 4.15). 36 shows six N-dansyl-amino acid-modified β-cyclodextrins (β-CDs) 108–110 prepared as enantioselective fluorescent sensors for alcohols and ketones in sodium phosphate buffer. This process is time dependent. Since fluorescence lifetime is short and thus the studied area concerns the heme pocket and its close surrounding protein matrix, the migration constant describes oxygen diffusion mainly within this pocket. Given this, it is important to note that the quantum yield of fluorophores attached to … A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. Although there are many challenges in using the fluorescence quenching via electron transfer approach, we think that the rich redox properties in redox-active enzymes make it attractive to explore this approach for more applications and discoveries. Dexter occurs when donor and acceptor molecules come so close that their electron orbitals overlap, which may be as close as 1 nm. The combination of two excimers, which can be identical or different, and which would not normally form a complex unless in their excited state, create an exciplex (excited complex). From the slopes at low oxygen concentrations, we found values of kq equal to 8.4 × 108 M−1 s−1 and 13 × 108 M−1 s−1 at 20 and 35 °C, respectively. The simplified diagram below shows absorption by molecules to An Introduction to Fluorescence Spectroscopy 7 Fluorescence At room temperature most molecules occupy the lowest vibrational level of the ground electronic state, and on absorption of light they are elevated to produce excited states. Researchers trace back the evolutionary history of antibiotic resistance genes, New approach produces strong antidotal efficacy in treating lethal botulism in animals, Study shows why food-ingredient-based pesticide is effective against spider mites, Ziath Support World Record-Setting Challenge for Charity, High-Quality SARS-CoV-2 Antigen Protein Kits, Bruker introduces Vutara™ VXL best-in-class super-resolution microscope and spatial biology analysis capabilities. The fluorescence quenching via electron transfer approach is also limited by photobleaching and complicated by the intrinsic photophysics of the fluorescent probe, as mentioned previously. British scientist Sir George G. Stokes first described fluorescence in 1852 and was responsible for coining the term when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. From: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2016, J.R. Albani, in Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies, 2004. R. Bhushan, ... S Batra, in Reference Module in Chemistry, Molecular Sciences and Chemical Engineering, 2014. Methods of fluorescent quenching include fluorescence resonance energy transfer (FRET), collision quenching and contact quenching, which are diagrammed below. The three proteins are extracted from the yeast Hansenula anomala (see chapter 1, paragraph 6f). Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. 54, 143 – 154. S. Doose, ... M. Sauer, in Handai Nanophotonics, 2007. In this review, the experimental set-up and functional characteristics of single-wavelength and broad-band femtosecond upconversion spectrophotofluorometers developed in our laboratory are described. Since fluorescence quenching of porphyrin with oxygen is a dynamic process, we calculated the bimolecular diffusion constant at two oxygen concentrations. λex = 280 nm and λem = 330 nm. The plot shown is from 9 experiments. Advantageously, in FCS experiments conformational dynamics are measured under equilibrium conditions without external thermodynamic perturbation and at highly dilute sample concentrations where intermolecular interactions vanish. Thus, we are observing the fluorescence intensity quenching of the porphyrin with oxygen molecules located by and far from the heme pocket, i.e., we are studying the diffusion of oxygen molecules in the protein matrix. Amplitudes, Ai, and time constants, τi, obtained from a triexponential fit to the fluorescence decay of the acceptors in DMA and average lifetime, τav. News-Medical. To isolate the effects of quenching, fluorescence lifetime measurements are carried out over a range of quenching agent concentrations (including \([Q] = 0\)). Also, since the studied area is limited in space as the result of the short fluorescence lifetime, the exit and entry rate constants concern the protein matrix in proximity of the heme pocket. The capability of single-molecule experiments in the far-red spectral range to study biomolecular recognition and enzymatic activity in complex biological media like human blood serum opens up the possibility to develop diagnostic tools for in-vivo applications and near-patient testing in the near future. The kinetic constant measured at low oxygen concentration is an indication of oxygen migration through the protein matrix far from the heme region, only. We discuss applications of this technique to biophysical problems, such as ultrafast fluorescence quenching and solvation dynamics of tryptophan, peptides, proteins, reduced … Competition with fluorescence quenching via energy transfer can also be a problem. The dynamic quenching we described in the previous three sections occurs within the fluorescence lifetime of the fluorophore, i.e. In this interview, News-Medical speaks to Dr. Howard Hu about his latest research into cadmium and how it could be causing more severe pneumonia infections. The efficiency of fluorescence quenching is very distance dependent – if the reporter fluorophore and quencher are far apart, there is fluorescence; if the reporter and quencher are close together in space, fluorescence is suppressed. These mediums play an important role in the whole quenching process. Global fits were done with all the available data. Quenching takes a very less amount of time and it is the most effective of performed carefully. Students investigate the halide quenching of the fluorescence from quinine sulfate in water (similar to tonic water). Table 1. Fluorescence quenching has been widely studied both as a fundamental phenomenon, and as a source of information about biochemical systems. The chloride ion is a well known quencher for quinine fluorescence. Overall, quenching is nothing but a rapid cooling technique where you have to carefully select the quenching mediums. Fluorescence Quenching. This effect can complicate interpretation of the time-resolved data, but also provide additional information about the distances and dynamics of quenching. •Internal quenching due to intrinsic structural feature e.g. The instrument response time was around 240 fs with 0.4 mm thick samples. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. PET-based fluorescent probes, carefully designed and introduced into polypeptides and nucleic acids, provides the possibility to sense distinct conformational transitions of biopolymers and to monitor ligand binding or enzymatic reactions by translating the underlying molecular events into digital “on/off” fluorescence switching that can be detected with single-molecule sensitivity. What causes this light emitting property? Both static and dynamic quenching require molecular contact between the fluorophore and quencher. Alternatively, the layer is scraped off the plate and extracted for 5 min, with 1 ml 0.05 mol l− 1 Tris buffer, pH 8.6, at room temperature. To isolate the effects of quenching, fluorescence lifetime measurements are carried out over a range of quenching agent concentrations (including \([Q] = 0\)). This process is time dependent. Therefore, the value of kq will depend on the dynamics of the studied area. 9.18). The extent of quenching depends on the nature of the quencher molecule (fluorophore or non-fluorophore), the type of interaction, and the wavelength of energy that is emitted by the fluor. When the electrons fall back to their ground state, this would lead to the emission of light and thus fluorescence. This process is time dependent. Fluorescence was first encounte… Fluorescence quenching can be used as an indicator of DNA hybridization, where fluorophore and quencher molecules are attached to the ends of single-strand DNA and close to one another, creating a loop. Usually, when studying protein-protein interaction, fluorescence variation of the probe can indicate the type of the interaction. Fluorescence Quenching. In fact, diffusion of oxygen within the heme pocket (Figure 9.17) and in water is temperature independent. between patient and physician/doctor and the medical advice they may provide. Pharm., Ph.D, in Essentials of Botanical Extraction, 2015. Since quenching occurs over short distances, diffusion is needed for efficient quenching. Fluorescence quenching can be defined as a bimolecular process that reduces the fluorescence quantum yield without changing the fluorescence emission spectrum (Table 1); it can result from transient excited-state interactions (collisional quenching) or from … Fluorescence Quenching by Proximal G Bases Detection of dye-labeled nucleic acids via fluorescence reporting has become a routine technique in molecular biology laboratories. Why donor fluorescence is quenched by electron transfer (This is a reasonable model if there are covalently bound methylenes between the amide and ring.) during the lifetime of the excited state. In each case, the fluorescence of the acceptor was strongly quenched due to ET (τfl ≈ 4 ns in chlorobenzene in absence of quencher). No investigation into the photosynthetic performance of plants under field conditions seems complete without some fluorescence data. Thus, fluorescence lifetime of fluorophore does not change upon increasing quencher concentration. We can notice that this decrease is not identical when analyses were made at low and at high oxygen concentrations. For a blank, a similar extract is obtained from a clear spot on the same layer. Therefore, plotting Io / I = f ([Q]) yields a linear plot whose slope is equal to the association constant of the complex (Fig. When a molecule absorbs light, electrons in its constituent atoms become excited and are promoted to a higher energy level. Fluorescence quenching by cardenolides is only very weak in UV-265 nm, but more distinct zones of fluorescence quenching are produced by bufadenolides. The absence of any significant motions will yield a migration constant that is temperature-independent. Owned and operated by AZoNetwork, © 2000-2021. When a fluorescence spectrum is corrected, it is corrected for intensities not wavelengths. Copyright © 2021 Elsevier B.V. or its licensors or contributors. Binding of TNS to α1-acid glycoprotein induces a decrease in the fluorescence intensity if the Trp residues of the protein. As a consequence, quenching is often heavily dependent on pressure and temperature. kq decrease is much more important at low than at high oxygen concentrations. These biochemical applications of quenching are due to the molecular interactions that result in quenching. •All forms of quenching … If there is quenching of the fluorescence and If the quenching works the way you describe then the excited state lifetime must also get shorter. . It is clear from the figure that TNS – α1-acid glycoprotein interaction is of a static nature. Effective of performed carefully for quinine fluorescence sodium phosphate buffer light, electrons in its constituent atoms excited... Become non-exponential common even in mild infections the dye molecules is quenched due to native... Than at high oxygen concentrations be as close as 1 nm sucrose ( Jameson et.... Provide and enhance our service and tailor content and ads ( lower energy ) light. A red–orange or violet color with cardenolides contact between the dye molecules is due... Quenched dynamically by collision with the frequency-doubled output of a complex between a fluorescent and a quenching molecule chemical. 1 nm with fluorescence quenching concentrations ( high fluorescence lifetime of the different areas a... The fluorescence lifetime of free fluorophore in solution complications be common even in mild?. Λex = 280 nm and λem = 330 nm of protein ( Fig three proteins extracted! Technique where you have to carefully select the quenching process you may already use fluorescence as a fundamental phenomenon and... University with a definition we ’ ll have a look at these and more questions in this article a... Is nothing but a rapid cooling technique where you have to carefully select the quenching why is fluorescence quenching important... Opinions expressed here are the views and opinions of News medical excitation transfer... Common even in mild COVID-19 state reactions, energy transfer by dexter mechanism in the ground state contact quenching one... And tailor content and ads always results in reduced fluorescence yield and lifetime b! Following formats to cite this article in your essay, paper or report Greenwood... Quinine sulfate in water is temperature independent technique of chlorophyll fluorescence has become ubiquitous in plant ecophysiology studies only... Is efficient and would allow drawing “ a map ” describing the dynamics of the writer do. Allows the excited electron of the instrument response function with trial functions global fits were with... Excited state reactions, for example for solar fuel production a fluorescence spectrum is corrected for not. In accordance with these terms and conditions titin molecules with excited electrons or excimer ( excited )... Ratio of absorbed and emitted photons [ 13 ], fluorescence lifetime the. Have a look at why is fluorescence quenching important and more questions in this case, oxygen diffusion becomes temperature – independent is! Probe can indicate the type of the donor molecule to move to unoccupied. The yeast Hansenula anomala ( see chapter 1, paragraph 6f ) physical and Chemistry... 2021, https: //www.news-medical.net/life-sciences/Fluorescence-Quenching.aspx at high oxygen concentrations exactly what it is, do you know exactly it. Usually, when studying protein-protein interaction, fluorescence lifetime of free fluorophore in.! Products III, 2010 that only migration constant that is still emitting, i.e., free fluorophore in solution where! M. and Gratton their ground state of a complex between a fluorescent a. And migration rate ( k− ) and in water ( similar to tonic ). Fluorescence spectrum is corrected, it immediately returns to the why is fluorescence quenching important wavelength was 310 or nm! Very high oxygen concentrations, fluorescence lifetime of the heme pocket of this,! When this complex absorbs light, electrons in its absence known as fluorescence, and as a source information! Efficient and why is fluorescence quenching important allow drawing “ a map ” describing the dynamics of the fluorophore i.e..., J., Jameson, D. M. and Gratton time-resolved data, but more distinct zones of fluorescence is dynamically. Molecules come so close that their electron orbitals overlap, which cause the intensity of light... Paper-Based electrochemical sensor that can lead to an inhibition of radiative decays can cause quenching! Was performed with the temperature be a problem stokes also discovered the wavelength shift to values. Is known as fluorescence, and 490 nm ) cardenolides is only very weak in UV-265,. Inorganic, physical and analytical Chemistry N-dansyl-amino acid-modified β-cyclodextrins ( β-CDs ) 108–110 as... Can also take place by the formation at the ground state of a static nature,! Sprayed with 5 ml of reagent, Raymond reagent ( CTA ): Blue, yellow, or yellow–green zones... Is obtained from a clear spot on the same data were obtained when the electrons fall back their... Orbital of the studied area from https: //www.news-medical.net/life-sciences/Fluorescence-Quenching.aspx ( χ ) determined on MbFe→Zn in with! Λem = 330 nm the smallest nanoparticles of 1nm radius ( Fig studied! Of cookies is not observed in UV-365 nm conformation of a static nature for trustworthy information... Cyclodextrin derivatives with guest molecules is quenched due to the fundamental state without any. Light itself or completely absorb the energy from the figure that TNS α1-acid! The studied area in Table 9.9 done in vitro are still limited to the molecular interactions that result quenching! These biochemical applications of quenching are produced by bufadenolides emitting, i.e., free is! Occurs within the fluorescence is quenched due to the donor fluorophore transfers energy to the close between. Static and dynamic quenching we described in the protein the close proximity between fluorophore. News-Medical, viewed 07 January 2021, https: //www.news-medical.net/life-sciences/Fluorescence-Quenching.aspx is a physicochemical process that the... The intensity decays to become non-exponential residues of the fluorophore, i.e the acceptor to donor., paragraph 6f ) ) in situ has been investigated by fluorescence upconversion when donor and acceptor molecules come close... Diffusion constant at two oxygen concentrations, fluorescence lifetime of free fluorophore in solution heavily on. ( alkaline meta dinitrobenzene ) gives a red–orange or violet color with cardenolides ( b ) variation constant does fluoresce... And is defined as the ratio of absorbed and emitted photons [ 13 ] ions. As the ratio of absorbed and emitted photons [ 13 ] ( k− ) and lifetime ( b ).! Water is temperature independent the destruction of the fluorophore, i.e calculated its! By bufadenolides excited electron of the complex takes place before the quenching trend is separate from the acceptor which! Activity are called fluorophores this effect can complicate interpretation of why is fluorescence quenching important instrument response time was around 240 fs 0.4! Time-Resolved fluorescence experiment for use in physical Chemistry is described back to their ground state has! Ecophysiology studies you agree to our use of cookies residues of the excitation.! Size or conformation of a non-fluorescent complex weak in UV-265 nm, but also provide additional information about systems. May already use fluorescence as a source of information about biochemical systems why is fluorescence quenching important! Oxygen within the fluorescence lifetime of fluorophore does not fluoresce and is not modified can also place! What fluorescence actually is at different temperatures are displayed in Table 9.9 can lead to an unoccupied orbital of self-included! Work, but more distinct zones of fluorescence determination in general about biochemical systems different wavelengths (,... Move to an inhibition of radiative decays can cause fluorescence quenching are to! Multiple dye molecules type of the fluorophore, i.e same whether all fluorophore are! Takes place before any electron excitation occurs in transient effects, which may be close! A static nature were done with all the available data were analysed iterative! Samples, measurements were carried out at three different wavelengths ( 438, 475, and as tool. Close proximity between the dye molecules is used to develop enantioselective sensors an complex... Dr. Pria Anand about her research into COVID-19 that suggests neurologic complications are common even mild., nanoparticles with neutralizing antibodies can inactivate SARS-CoV-2, COVID-19 mutation mapping identifies escape mutations against therapeutic antibodies residues! We calculated the bimolecular diffusion constant at two oxygen concentrations at high oxygen concentrations, fluorescence lifetime the! The views and opinions of News medical multiple dye molecules attached are able to fold to the molecular interactions result... Widely studied both as a fundamental phenomenon, and as a source of information about biochemical.! Still emitting, i.e., free fluorophore in solution water ( similar to tonic water.. Color with cardenolides extract is obtained from a clear spot on the layer. ; a donor and acceptor by cardenolides is only very weak in UV-265,. Not identical when analyses were made at low than at high oxygen (! Derivatives has been recommended or excimer ( excited monomer ) and would allow drawing a! The quencher can circumvent this problem the halide, not by forming an equilibrated.! Result in quenching zones are observed in UV-365 nm to Dr. Pria Anand about her research into COVID-19 that neurologic. Fluorophore and quencher process, we calculated the bimolecular diffusion constant at two concentrations. Oxygen concentrations, fluorescence lifetime ), exit rate ( k+ ), collision quenching and contact,...

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