Fluorescence multidimensional

Electrodriven separation techniques are destined to be included in many future multidimensional systems, as CE is increasingly accepted in the analytical laboratory. The combination of LC and CE should become easier as vendors work towards providing enhanced microscale pumps, injectors, and detectors (18). Detection is often a problem in capillary techniques due to the short path length that is inherent in the capillary. The work by Jorgenson s group mainly involved fluorescence detection to overcome this limit in the sensitivity of detection, although UV-VIS would be less restrictive in the types of analytes detected. Increasingly sensitive detectors of many types will make the use of all kinds of capillary electrophoretic techniques more popular. 

Even the fact that some species do not fluoresce may be used as a selectivity tool. Computers and modem electronics have made multidimensional measurements possible on a hitherto unprecedented scale and this approach has been one of the most effective ways to achieve high selectivity in luminescence measurements. Many ingenious techniques have recently become available for utilizing the multiple luminescence variables and these have made luminescence measurements a routine and valuable tool in almost all areas of experimental science. 

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

Multidimensional Fluorescence Imaging for Non-Invasive Tracking of Cell Responses 623... 

In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1993a). To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977 May and Wise 1984 Wise et al. 1993a, 1993b), consists of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their alkyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. 

The aim of this review was to summarize those aspects of fluorescence spectroscopy that may have value for solving problems in food science and technology. The techniques described, which are mainly based on front-face fluorescence spectroscopy coupled with multidimensional statistical methods, have been illustrated by examples taken from the literature and the work done in our laboratory. Although fluorescence spectroscopy is a technique whose theory and methodology have been extensively exploited for studies of both chemistry and biochemistry, the utility of fluorescence spectroscopy for molecular studies has not yet been fully recognized in food science. Fluorescence spectroscopy has the same potential to address molecular problems in food science as in the biochemical science field, because the scientific questions that need to be answered are closely related. We hope that this coverage will introduce a novel class of techniques in the emulsion and gel fields. 

M.W. Urban and T. Provder, Multidimensional Spectroscopy of Polymers. Vibrational, NMR, and Fluorescence Techniques ACS Symposium Series No. 598, American Chemical Society, Washington, 1X7 (1995). 

One often finds that when high resolution separation schemes are utilized, other techniques and disciplines must participate in the scheme of understanding and effectively utilizing the separation with subsequent identification of the resulting zones. A rigorous and often multidimensional detection scheme such as mass spectrometry and/or fluorescence is found both for the life science and industrial polymer applications. Other disciplines including informatics and statistics are often... 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Bastiaens, P. I. H., van Hoek, A., Benen, J. A., Brochon, J. C. and Visser, A. J. W. G. (1992). Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study. Biophys. J. 63, 839-53. 

C. D. Tran and R. J. Furlan, Acoustic-optic tunable filter as a polychromator and its application in multidimensional fluorescence spectrometry, Anal. Chem. 64, 2775-2782(1992). 

Selected entries from Methods in Enzymology [vol, page(s)] Application in fluorescence, 240, 734, 736, 757 convolution, 240, 490-491 in NMR [discrete transform, 239, 319-322 inverse transform, 239, 208, 259 multinuclear multidimensional NMR, 239, 71-73 shift theorem, 239, 210 time-domain shape functions, 239, 208-209] FT infrared spectroscopy [iron-coordinated CO, in difference spectrum of photolyzed carbonmonoxymyo-globin, 232, 186-187 for fatty acyl ester determination in small cell samples, 233, 311-313 myoglobin conformational substrates, 232, 186-187]. 

Duncan, R.R., Bergmann, A., Cousin, M.A., Apps, D.K., and Shipston, M.J. et al. 2004. Multidimensional time correllated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (Aim) to Detect Fret in Cells. J. Microsc. 215 1. 

An indication of the potential sensitivity of fluorometry is that single-molecule detection has been based almost exclusively on the use of fluorescent labeled compounds. In addition, fluorometric determinations can combine several parameters simultaneously ( multidimensional techniques), such as excitation and emission wavelengths, fluorescence lifetime, and polarization, providing additional specificity and versatility to the analytical measurements. 

Several configurations for the sensor are possible. An especially viable alternative would seem to be the competitive displacement of fluorescent label. Since this is an equilibrium, fouling or contamination of the surface should not alter the absolute result. Krull et al (75) have reported the reproducible immobilisation of a stable phospholipid membrane containing fluorophore in this context. Concurrent fluorescence polarisation measurements can offer the possibility of multidimensional analysis (76) and are in any case experiencing a rejuvenation of interest as a highly selective technique, when the effective molecular weight of the antibody is increased relative to the antigen, by immobilisation on a latex or metal particle (77)... 

The data shown below illustrates how the concept of multidimensional analysis might be used to devise a simultaneous fluorescence assay of warfarin and its major metabolite, 7-0H warfarin. 

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. 

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