Multidimensional fluorescence analysis systems

Multidimensional Fluorescence Analysis of Cyclodextrin Solvent—Extraction Systems... 

An added feature of total fluorescence techniques is found in systems whose emission and/or excitation wavelength maxima change with the formation of a complex. Multidimensional fluorescence analysis allows the analyst to detect the differences in sensitivity arising from changes in excitation and emission maxima of the analyte. In addition, the technique finds wide application in examining the corrected fluorescence spectra of clinical compounds and their derivatives. In studies involving biological... 

Multidimensional fluorescence analysis is well suited for routine qualitative separation and identification of complex mixtures of PAHs in environmental samples. The separation and extraction of the individual component spectra of PAH mixtures affords valuable information about these hazardous compounds. Such information may allow for a detailed study of the means to remove their influence from the environment. The differences in the intensity contributions from a mixture of PAHs in a multicomponent system could be related to the differences in concentrations of the individual components and their respective quantum efficiencies. The problem is exacerbated by the fact that most solutions containing mixtures of PAHs display broad fluorescence spectral bands at room temperature, resulting in significant overlap and limited specificity for multicomponent analysis. This makes the success of multidimensional fluorescence measurements for complete spectral resolution of a complex multicomponent mixture dependent upon the degree of spectral overlap among the individual components and their fluorescence... 

In this section, based on the methodology presented in the previous section, we describe multidimensional fluorescence imaging and its application to tracking cell responses. We developed the time- and spectrally-resolved fluorescence imaging system based on line illumination, which is capable of rapid acquisition of fluorescence intensities as a function of Em, x, and xy-positions. We applied it to the analysis of an induced plant defense response, that is, the accumulation of antimicrobial compounds or phytoalexins, in oat (Avena sativa). 

Warner and co-workers justify the elaborate optical/detection system and the time commitment required per analysis on the basis of the additional sensitivity available using fluorescence detection, and on the multidimensional CD information available. For applications in which two, or more CD active fluorophores may be present, the ability to provide both an excitation and emission FDCD profile for the sample may allow differentiation of the individual components without pre-separation. Replacement of the mechanical mechanism for prism movement with an electro-optical device may improve both the SNR and reduce the time required per sample. These improvements will greatly facilitate general application of this multidimensional approach to FDCD measurements. 

Summarizing this section, we developed the time-gated excitation-emission matrix spectroscopic system and applied it to the decomposition of a mixed solution of a number of fluorescent dyes. We demonstrated that our approach, which was based on unique optical configuration, efficient acquisition of a multidimensional data set, and decomposition of unknown fluorescent components by using the PARAFAC model, was effective for the analysis ofunknown multi-component targets. 

The acquisition of a single-wavelength fluorescence spectrum of a complex multicomponent mixture is more tedious for many analytical applications that involve the measurement of some essential intermediate components or transient species. The determinations of such systems become more critical if the spectral information for those species is important to the understanding and elucidation of the properties of the final product. Similarly, this is true for chemical measurements of components whose fluorescence intensity decreases continuously with time. A rapid multidimensional data-processing technique becomes exceedingly important in the analysis of these species in a chemical reaction. 

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