Fluorescence polarisation spectroscopy

Fluorescence polarisation spectroscopy is still very much used to probe the rotational dynamics of single molecules, either on surfaces or in solution [152]. In bioa-nalytical assays the fluorescence emission intensity is measured as a function of rotational speed. When a solution of fluorophores is excited with polarised light, the fluorophores selectively absorb those photons that are parallel to the transition moment of the fluorophore, resulting in photoselective excitation. The fluorophore molecules rotate to varying extents during the fluorophore lifetime. If the fluores-... 

Litvinenko KL, Webber NM, Meech SR (2001) An ultrafast polarisation spectroscopy study of internal conversion and orientational relaxation of the chromophore of the green fluorescent protein. Chem Phys Lett 346 47-53... 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Fluorescence Polarisation coupled with confocal spectroscopy... 

In Table 3 the orientation information which can be obtained from these various structural techniques is summarised. This table also shows the part of the molecular structure which is being characterised, and some of the theoretical and experimental limitations of each method. A further technique, that of polarised fluorescence has been added. This technique is exactly analogous in its orientation aspects to Raman spectroscopy. The distinction between the two techniques lies in the fact that in the Raman effect, the lifetime of the process is of the order of the vibrational period ( 10 s) whereas fluorescence occurs after much longer occupancy of the transition state ( 10 s). 

Laser-Raman spectroscopy Molecular bond directions can be specific to crystalline or non-crystalline regions, parts of structure cos 0, cos 0, cos Ocos , etc. (hyper-Raman might give higher moments) To relate polarisability changes to directions in molecules may not be as straightforward as assuming a bond direction correspondence Transparent specimens, with little or no fluorescence required... 

Polarisation studies are common in fluorescence spectroscopy. In the more expensive instruments internal polarisers may be automatically switched between horizontal and vertical alignments, otherwise polarisers placed in the sample compartment require manual switching. Plastic polarisers are relatively inexpensive but will absorb and be bleached by hght below about 300 nm and so are unsuitable for UV work. 

In alcohols, the solvation times agree remarkably with the reorientation times of the free alcohol molecules see below, Section 5.3.3. In non-polar solvents, other secondary-sphere solvent molecules may quickly undergo considerable polarisation, as is shown by the difference between optical-absorption and fluorescence-emission spectra measured a few picoseconds after initiation these spectra show maxima at different wavelengths (the Stokes shift ), which can be accounted for by electrostatic models. See Section 6.3.4 below, and G.R. Fleming, Chemical Applications of Ultrafast Spectroscopy, Clarendon Press, Oxford, 1986, pp. 166, seq. 

As a result of the high polarisability of C—S and S—S bonds, Raman spectroscopy is especially suitable for studying sulfur vulcanisation of elastomers. However, conventional Raman studies of elastomers are limited on account of sample fluorescence (often due to impurities). 

As in absorption spectroscopy, instrumental polarization effects can yield unwanted artefacts and therefore it is appropriate to introduce depolarizers into the optical path before and after the sample if the aim is to monitor the true unpolarized fluorescence spectrum. Unfortunately, this will reduce the light levels com-mensurately and is therefore frequently not pursued. Other methods, involving the use of polarizers set at magic angles to minimize some unwanted polarisation effects have been devised, but are even less frequently employed. 

London, Chapman Hall, 1997, p.181-233. 9 INFRARED DICHROISM, POLARISED FLUORESCENCE AND RAMAN SPECTROSCOPY... 

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