Quantum yield common fluorophores

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]. 

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],... 

A high degree of sensitivity and selectivity can be obtained with certain biomolecules by the chemical attachment of fluorophores. The most common fluorescent derivatization reagents include fluorescamine, dansyl chloride, pyridoxal, pyridoxal 5-phosphate, dansyl hydrazine, and pyr-idoxamine. Such derivatization procedures can be used to enhance the fluorescence of compounds with low quantum yields as well as impart fluorescent properties to compounds that do not fluoresce naturally. 

Another related phenomenon that results in a lower quantum yield than expected is called concentration quenching. This can occur when a macromolecule, such as an antibody, is heavily labeled with a fluorophore, such as fluorescein isothiocyanate. When this compound is excited, the fluorescence labels are in such close proximity that radiationless energy transfer occurs. Thus, the resulting fluorescence is much lower than expected for the concentration of the label. This is a common problem in flow cytometry and laser-induced fluorescence when attempting to enhance detection sensitivity by increasing the density of the fluorescing label. 

Solvent polarity and the local environment have profound effects on the emission spectra of polar fluorophores. These effects are the origin of the Stokes shift, which is one of the earliest observations in fluorescence. Emission spectra are easily measured, and as a result, there are num ous publications on emission spectra of fluoropho-res in different solvents and when bound to proteins, membranes, and nucleic acids. One common use of solvent effects is to determine the polarity of the probe binding site on the macromolecule. This is accomplished by comparison of the emission Spectra and/or quantum yields of the fluorophore when it is bound to the macromolecule and when it is dissolved in solvents of different polarity. However, there are many additional instances where solvent effects are used. Suppose a fluorescent ligand binds to a protein. Binding is usually accompanied by a spectral shift due to the different environment for the bound ligand. Alternatively, the ligand may induce a spectral shift in the intrinsic or extrinsic protein fluorescence. Additionally, fluorophores often display spectral shifts when they bind to membranes. 

The modular construction of fluorescent supramolecular proton sensors allows the use of a great variety of fluorophore and receptor units. The majority of the supramolecular fluorescent proton sensors possess an amine component as the receptor. The simplest and most common cases are those having an aromatic hydrocarbon as the fluorophore, but systems based on coordination compounds have also been proposed. For example Grigg and Norbert have synthesized supramolecular species where amine units are covalently linked to Ru(bpy)3 (see, e.g., 9). In neutral solution the amine units are unprotonated and the luminescence of the Ru(bpy)3 moiety is completely quenched when AG for electron transfer is negative. Protonation of the amine groups (pKa=2.4 for 9) leads to the retrieval of the luminescence. The luminescence maxima and quantum yields of the protonated forms, however, are different from those of free Ru(bpy)3 " , presumably because of the presence of the positive charges in the vicinity of the luminophore. 

Photobleaching Photobleaching is the photochemical destruction of a fluorophore, which is common to organic dyes and in time-lapse microscopy. It means that high-energy excitation light renders a fluorophore not to fluoresce or with a lower quantum yield. 

Either, or both, excitation source modulation frequency or detector response can limit instrument capabilities. Most standard commercial instruments have modulation frequencies up to a maximum of a few hundred MHz and are best suited for relatively long-lived fluorophores with high quantum yields. The excitation source is typically a modulated LED or laser diode, or for a wide wavelength range, light from a continuum steady-state source, such as a Xe arc lamp modulated with an electro-optical cell, such as a Pockels cell detection is typically a fast photomultiplier, or multichannel plate photomultiplier. Polarisers are commonly used accessories. Such an instrument is ideally suited for lifetimes of a few ns, but will also measure, albeit with lower precision, lifetimes in range of lOO s ps. GHz modulation frequencies are obtained with mode locked lasers with a fast multichannel plate photomultiplier and these allow lifetime measurements in the range of tens of ps. 

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