Extrinsic fluorescent probes selecting

One can employ linearly polarized light to excite selectively those fluorophores that are in a particular orientation. The difference between excitation and emitted light polarization changes whenever fluorophores rotate during the period of time between excitation and emission. The magnitude of depolarization can be measured, and one can therefore deduce the fluorophore s rotational relaxation kinetics. Extrinsic fluorescence probes are especially useful here, because the proper choice of their fluorescence lifetime will greatly improve the measurement of rotational relaxation rates. One can also determine the freedom of motion of the probe relative to the rotational diffusion properties of the macromolecule to which it is attached. When held rigidly by the macromolecule, the depolarization of a probe s fluorescence is dominated by the the motion of the macromolecule. 

The selection of extrinsic fluorescent probe is driven by the consideration of which biological macromolecule or lipid is to be labelled, the requirement for compatibility between the intended fluorescent probe (in terms of solubility in water, pH sensitivity and so on) and the properties of the molecule to be labelled. Also, choice of the fluorescent probe should be consistent with experimental objectives. For instance, FRET experiments require that extrinsic donor and acceptor fluorophores should be properly matched for their capacity to participate in the FRET effect (see Section 4.5.4). 

As nonconjugated and nonaromatic polymers are nonfluorescent, studies of their phase behavior using fluorescence spectroscopy require the use of extrinsic fluorescent labels [38-40]. These can be either selectively dissolved into the polymer phases [41] or, most commonly, attached covalently to the polymer chains [42-47]. A criticism that is often made of using extrinsic fluorescent probes is the possible local perturbation induced by the probe itself on the nanoenvironment to be probed. In order to minimize such perturbation, the size and shape of the probe should be chosen so as to cause the minimum possible perturbation on the probed region. 

A variety of extrinsic fluorophores can be attached to proteins to serve as fluorescence probes. These can be selected to maximize sensitivity and to avoid contamination (i.e., by moving to longer absorption and emission wavelengths) from other absorbing components. With both intrinsic and extrinsic fluorescence probes, the method focuses only on these probes sites, which might be as few as a single site on a protein. 

Biological Systems - The sensitivity of luminescence techniques and the use of extrinsic and intrinsic fluorescence probes have found considerable applications in biological research. Only a selection of such studies have been selected for citation. 

The usual experimental technique is to observe the fluorescence of a probe molecule incorporated into the system under investigation and make the assumption that the motion of the probe reflects that of its environment. Probe molecules may be divided into two cathodes intrinsic probes (such as fluorescent amino acid residues naturally occurring in a protein under investigation) and extrinsic probes either bound chemically to the macromolecule or simply dispersed in the stem. The criteria that need to be applied when selecting suitable candidates for probe molecules have been discussed by Stryer Ideally the probe should... 

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