Quantum yield, fluorophore

Gryczynski, L, et al. (2002) The CFS Engineers the Intrinsic Radiative Decay Rate of Low Quantum Yield Fluorophores. J Fluoresc 12 11-13. 

The fluorescence amplification provided by the plasmonic nanostructures has been shown to be applicable to many fluorophores. Hence fluorophores currently employed in assays would still be suitable. However, the use of low quantum yield fluorophores would lead to much larger fluorescence enhancements (i.e. 1 / Qo) and could significantly reduce unwanted background emission fi om fluorophores distal fi om the metallic surface. 

As already pointed out an additional radiative rate results in an increase in the total radiative decay rate. If a dye has a high quantum yield near one, the additional radiative decay rate cannot substantially increase Ae quantum yield. The more interesting case is for low quantum yield fluorophores. In this case the enhancement can be boosted to near one and thus an overall higher fluorescence amplifications is obtained. This suggests the emission from weakly fluorescent substances can be increased if they are positioned at an... 

Overall, the significant Stokes shift of 100 nm and the good quantum yields make the coumarin dye a powerful fluorescent probe for nucleic acids assays or cell biology. The postsynthetic click chemistry makes this fluorophore readily accessible for fluorescent labeling of nucleic acids. 

The amine-reactive 5-(dimethylamino)naphthalene-l-sulfonyl (dansyl) chloride 28 [80] and related fluorophores [81, 82], as well as the 5-((2 aminoethyl)amino) naphthalene-1-sulfonic acid (EDANS) 29, are included in the naphthalene fluorophore family. Derivatives of the latter, such as compound 30, exhibit a Lm.ix/ Lem 336/520 nm, molar absorptivity (e) of 6.1 x 103 M-1 cm-1, and a fluorescent quantum yield of 0.27 in water [83], The use of EDANS is particularly interesting in FRET experiments [84, 85]. Furthermore, 4-amino-3,6-disulfonylnaphthalimides (e.g., Lucifer yellow 31), associated to a longer absorption (Lmax 428 nm) [86] are suitable polar tracers [87]. 

Fluorophores containing 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as a core skeleton are commonly designated as BODIPY fluorophores. Due to their useful photophysical properties including high fluorescence quantum yields, high molar absorption coefficient, narrow absorption and emission band width, and their high photostability [50], BODIPY dyes are proven to be extremely versatile and useful in many biological applications Fig. 11 [68]. 

As seen from (1) and (2), intermolecular processes may reduce essentially the lifetime and the fluorescence quantum yield. Hence, controlling the changes of these characteristics, we can monitor their occurrence and determine some characteristics of intermolecular reactions. Such processes can involve other particles, when they interact directly with the fluorophore (bimolecular reactions) or participate (as energy acceptors) in deactivation of S) state, owing to nonradiative or radiative energy transfer. Table 1 gives the main known intermolecular reactions and interactions, which can be divided into four groups ... 

One of the most popular applications of molecular rotors is the quantitative determination of solvent viscosity (for some examples, see references [18, 23-27] and Sect. 5). Viscosity refers to a bulk property, but molecular rotors change their behavior under the influence of the solvent on the molecular scale. Most commonly, the diffusivity of a fluorophore is related to bulk viscosity through the Debye-Stokes-Einstein relationship where the diffusion constant D is inversely proportional to bulk viscosity rj. Established techniques such as fluorescent recovery after photobleaching (FRAP) and fluorescence anisotropy build on the diffusivity of a fluorophore. However, the relationship between diffusivity on a molecular scale and bulk viscosity is always an approximation, because it does not consider molecular-scale effects such as size differences between fluorophore and solvent, electrostatic interactions, hydrogen bond formation, or a possible anisotropy of the environment. Nonetheless, approaches exist to resolve this conflict between bulk viscosity and apparent microviscosity at the molecular scale. Forster and Hoffmann examined some triphenylamine dyes with TICT characteristics. These dyes are characterized by radiationless relaxation from the TICT state. Forster and Hoffmann found a power-law relationship between quantum yield and solvent viscosity both analytically and experimentally [28]. For a quantitative derivation of the power-law relationship, Forster and Hoffmann define the solvent s microfriction k by applying the Debye-Stokes-Einstein diffusion model (2)... 

Molecular rotors are useful as reporters of their microenvironment, because their fluorescence emission allows to probe TICT formation and solvent interaction. Measurements are possible through steady-state spectroscopy and time-resolved spectroscopy. Three primary effects were identified in Sect. 2, namely, the solvent-dependent reorientation rate, the solvent-dependent quantum yield (which directly links to the reorientation rate), and the solvatochromic shift. Most commonly, molecular rotors exhibit a change in quantum yield as a consequence of nonradia-tive relaxation. Therefore, the fluorophore s quantum yield needs to be determined as accurately as possible. In steady-state spectroscopy, emission intensity can be calibrated with quantum yield standards. Alternatively, relative changes in emission intensity can be used, because the ratio of two intensities is identical to the ratio of the corresponding quantum yields if the fluid optical properties remain constant. For molecular rotors with nonradiative relaxation, the calibrated measurement of the quantum yield allows to approximately compute the rotational relaxation rate kor from the measured quantum yield [Pg.284]

The amount of fluorescence emitted by a fluorophore is determined by the efficiencies of absorption and emission of photons, processes that are described by the extinction coefficient and the quantum yield. The extinction coefficient (e/M-1 cm-1) is a measure of the probability for a fluorophore to absorb light. It is unique for every molecule under certain environmental conditions, and depends, among other factors, on the molecule cross section. In general, the bigger the 7c-system of the fluorophore, the greater is the probability that the photon hitting the fluorophore is absorbed. Common extinction coefficient values of fluorophores range from 25,000 to 200,000 M 1 cm-1 [4],... 

The quantum yield (Q) represents the ratio between the number of photons absorbed and photons emitted as fluorescence. It is a measure of brightness of the fluorophore and represents the efficiency of the emission process. The determination of absolute quantum yield for a fluorophore is experimentally difficult. Therefore, usually relative quantum yield values are determined. To measure the relative quantum yield of a fluorophore, the sample is compared to a standard fluorophore with an established quantum yield that does not show variations in the excitation wavelength [5, 6]. 

Molecular rotors are fluorophores characteristic for having a fluorescent quantum yield that strongly depends on the viscosity of the solvent [50], This property relies on the ability to resume a twisted conformation in the excited state (twisted intramolecular charge transfer or TICT state) that has a lower energy than the planar conformation. The de-excitation from the twisted conformation happens via a non-radiative pathway. Since the formation of the TICT state is favored in viscous solvents or at low temperature, the probability of fluorescence emission is reduced under those conditions [51]. Molecular rotors have been used as viscosity and flow sensors for biological applications [52], Modifications on their structure have introduced new reactivity that might increase the diversity of their use in the future [53] (see Fig. 6.7). 

Fluorophore (M em ) Excitation wavelength (nm) Emission wavelength (nm) Solvent Quantum yield... 

Maximal overlap between the fluorescent emission spectra of the donor and the fluorescent absorption spectra of the acceptor is important for efficient FRET. Since the donor can be directly excited, even fluorophores with mediocre quantum yield may be used. However, the success of a ratiometric FRET approach will strongly depend on the quantum yield of the acceptor. While an acceptor with a lousy quantum yield may still be a good quencher of the donor fluorescence, it will not yield acceptor fluorescence to an appreciable extent. When calculating the ratio between donor and acceptor emission values, acceptors with a good quantum yield will therefore be preferred. 

Many fluorophores are sensitive to changes in the hydropho-bicity of the immediate environment. Therefore, bringing these fluorophores into a different environment may also produce a change in FRET, when a second fluorophore is affected by the emission change of the first. Fluorophores like Nile Red with changes of up to 100 nm when transferred from water to an aprotic organic solvent are principally suitable for such an approach [71], Molecular rotors have the characteristic of having a quantum yield that depends on the viscosity. Such dyes are formed by an electron donor unit and an electron acceptor unit that can rotate relative to each other upon photoexcitation with a behavior that depends on the viscosity of the environment. These dyes have been included in FRET probes for viscosity studies [53],... 

What is the donor/acceptor ratio in a given cell Again, this ratio cannot be directly derived because it concerns two quantities that stem from fluorophores with different properties (absorption coefficient, quantum yield, spectra) and that emit into two channels differing in gain, filters, and excitation intensity. Thus, the (overlap corrected) intensity of acceptors in channel A will be a factor k times that of donors in D, at equimolar concentrations,3 or ... 

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