Fluorescence multidimensional detection

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... 

This difficulty is easily avoided by fluorescence lifetime detection. By using the sequential recording capability of multidimensional TCSPC, the fluorescence transients can be directly observed. A simple setup for recording the nonphotochemical quenching is shown in Fig. 5.31. 

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. 

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. 

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. 

Relative protein quantitation is the basis of all types of differential proteome analyses. In the 2D-gel approach protein staining with either visible or fluorescent dyes provides a reliable and sensitive method to detect changes in protein expression or isoform abundance. In the multidimensional LC approach quantitation relies mostly on stable isotope labeling and ratios between light and heavy isotopomers are determined by MS or MS/MS at the peptide level. Labeling can be performed on the protein level by... 

We developed two kinds of multidimensional fluorescence spectroscopic systems the time-gated excitation-emission matrix spectroscopic system and the time- and spectrally resolved fluorescence microscopic system. The former acquires the fluorescence intensities as a function of excitation wavelength (Ex), emission wavelength (Em), and delay time (x) after impulsive photoexcitation, while the latter acquires the fluorescence intensities as a function of Em, x, and spatial localization (%-, y-positions). In both methods, efficient acquisition of a whole data set is achieved based on line illumination by the laser beam and detection of the fluorescence image by a 2D image sensor, that is, a charge-coupled device (CCD) camera. 

In this section, we will consider the use of multidimensional spectrometers as chromatography detectors. In multidimensional spectroscopy, the spectral intensity is a function of more than one spectral parameter. For exaiaple, detection by means of fluorescence is inherently t% o dimensional because the observed intensity is a function of two variables, wavelength of emission and travelength of excitation (15). Other examples of two dimensional spectroscopy aret MS IS ( ) and the v u ious types of two dimensional NMR ei riments (36). Appellof and Davidson... 

In the present paper we describe an apparatus for recording transient emission spectra that yields data which approach the ideal multidimensional case. We emphasize in the discussion the advantages of multichannel detection for transient emission data. We also briefly compare our approach to alternative methods for recording time and wavelength resolved fluorescence data on the picosecond time scale. 

Important applications of Af-PLS are in the area of multivariate calibrations for excitation/emission fluorescence spectrometry, for hyphenated analytical methods, such as HPLC/diode array detection and GC/MS, or for multidimensional separation techniques with or without coupling to spectroscopy. 

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... 

Many petroleum-based samples contain complex mixtures of PAHs, which are known or suspected carcinogens and mutagens. Multidimensional fluorescence analysis is suitable for the identification of fluorophores contained in oil samples. The basis of this technique is to employ a unique spectral fingerprint for the selective characterization and mapping of certain PAHs in the oil. Such a procedure is likely to provide a detection scheme for selective analysis of environmental samples for analytically important hazardous compounds. The primary goal will be to expand this technique to create a library containing... 

Another feature of multidimensional fluorescence is that it provides a good potential for a better spatial resolution, especially when using CCD detectors. The CCD detector is able to improve the detection of a fluorescent mixture of PAHs and provides a large dynamic range and reproducibility of spectral properties of the sample. 

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