Fluorescence spectral corrections

Obviousfy, this sensitivity versus X variation will distort the true fluorescence spectrum of the solute and lead to errors in quantitation, especially where spectral integration is performed. Superimposed on the PMT X sensitivity variation is a smaller effect of the light throughput dependency on X of the monochromator. 

There are three methods of generating spectral correction curves to accoimt for these sensitivity variations. One is to measure the fluorescence spectra of standard compounds whose true fluorescence spectra have been determined and reported (3). Then the sensitivity factors could be calculated by comparing the experimentally measured spectra with the reference spectrum. 

Rgura IX) The spectral sensitivity curve of a Hamamatsu R955 photomultiplier tube. 

A method that has shown to be quite satisfactory and relatively straightforward is described in Protocol 2. In this method a diffuser plate or an MgO scatterer plate is placed in the sample holder and then both ExM and EmM are scanned synchronously from 250-600 mn. This will produce a spectrum of the xenon arc source that has been distorted by the sensitivity variation of the emission detection system. The reflector is replaced by a concentrated sample of rhodamine B in a triangular cuvette. The cuvette is oriented so that the hypotenuse is on the face opposite to EmM. This is a similar configuration to the cell in the reference channel of the instrument. A red cut-oflf filter is placed in front of the entrance slit to EmM and Xem is set to 620 ran. Then ExM is scanned from 250-600 nm and the spectrum is recorded. This spectrum is a close representation of the true xenon lamp intensity profile. Often neutral density filters will have to be placed in the emission beam in order to prevent signal saturation of the PMT. The two curves thus generated can be compared and the correction fector curve, C(X), calculated  

If Fm(X) represents the measured uncorrected sample fluorescence spectrum then a corrected sample fluorescence spectrum can be obtained by a multiplication of C(X) and Fm(X) according to 

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In the following section, we discuss the fluorescence properties of AOM in terms of peaks observable in the spectrally corrected EEM data according to the scheme of Coble (1996), while at the same time attempting to reconcile the myriad of peak tables published in the past 20 years, with some speculation as to commonality among results. For the purpose of this chapter, we use the term peak in the context of spectroscopy practice as anything that exceeds the signal to noise of the background of the spectrum. The term... 

Holbrook, R.D., DeRose, P.C., Leigh, S.D., Rukhin, A.L., and Heckert, N.A. (2006). Excitation-emission matrix fluorescence spectroscopy for natural organic matter characterization A quantitative evaluation of calibration and spectral correction procedures. Appl. Spectrosc., 60(7), 791-799. 

Fluorescence units are often a term used to describe the intensity axis of the spectral plot (Resch-Genger, 2(X)7). In reality, there appears to be no standard definition of this term, nor any means to quantify it in an absolute radiometric meaning. There are so many effects, such as optical, sample, and instrumentation related, involved in fluorescence measurements that without a full and strict radiometric calibration fluorescence units are no more than some arbitrary scale of intensity. Huorescence units are therefore not directly comparable from instrument to instrument or from laboratory to laboratory. The situation becomes even more complex when thinking about excitation-emission matrices (EEMs) as the spectral position in excitation-emission space can be dramatically affected by the spectral correction used in both channels as well as the possibilities of signal saturations. These are important considerations to understand when reporting any fluorescence intensity data. 

Both instrument design and capabilities of fluorescence spectroscopy have greatly advanced over the last several decades. Advancements include solid-state excitation sources, integration of fiber optic technology, highly sensitive multichannel detectors, rapid-scan monochromators, sensitive spectral correction techniques, and improved data manipulation software (Christian et al., 1981 Lochmuller and Saavedra, 1986 Cabaniss and Shuman, 1987 Lakowicz, 2006 Hudson et al., 2(X)7). The cumulative effect of these improvements have pushed the limits and expanded the application of fluorescence techniques to numerous scientific research fields. One of the more powerful advancements is the ability to obtain in situ fluorescence measurements of natural waters (Moore, 1994). 

In addition to qualitative identification of the elements present, XRF can be used to determine quantitative elemental compositions and layer thicknesses of thin films. In quantitative analysis the observed intensities must be corrected for various factors, including the spectral intensity distribution of the incident X rays, fluorescent yields, matrix enhancements and absorptions, etc. Two general methods used for making these corrections are the empirical parameters method and the fimdamen-tal parameters methods. 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Calibration. In general, standards used for instrument calibration are physical devices (standard lamps, flow meters, etc.) or pure chemical compounds in solution (solid or liquid), although some combined forms could be used (e.g., Tb + Eu in glass for wavelength calibration). Calibrated lnstr iment parameters include wavelength accuracy, detection-system spectral responsivity (to determine corrected excitation and emission spectra), and stability, among others. Fluorescence data such as corrected excitation and emission spectra, quantum yields, decay times, and polarization that are to be compared among laboratories are dependent on these calibrations. The Instrument and fluorescence parameters and various standards, reviewed recently (1,2,11), are discussed briefly below. 

Shading correction is simply carried out by measuring the fluorescence of solutions of dyes that are spectrally similar to the donor and acceptor. The fluorescence of these reference images is then... 

The spectral overlap is an important quantity in radiationless energy transfer and migration, as we have seen in Eq. (32). It is equal to the integral of the corrected and normalized fluorescence intensity if (v) of the donor multiplied... 

When fluorochromes are combined in a multiparameter assay, it is almost always necessary to correct for signals from overlapping portions of emission spectra that have not been eliminated by optical filtration. This is accomplished through a spectral compensation procedure that is performed according to instrument-specific instructions, and involves the adjustment of detector voltages to electronically subtract extraneous fluorescence. 

Compounds 1,2,3,5,10,11,12,13,14 were dissolved in EPIP (diethyl ether, petroleum ether, isopropanol 5 5 2)whereas compounds 4,6,7,8,9,15 were dissolved in THF-DE (tetrahydrofurane, diethyl ether 1 1). These solvent mixtures can be frozen as glassy samples at 77 K. The absorption spectra were recorded on a standard spectrophotometer SF-10 or Beckman-5270. The measurements of fluorescence excitation and emission spectra were made with the aid of a spectrofluorometer SLM-4800 with automatic correction of spectral response. Fluorescence lifetimes were measured with the aid of a pulse fluorometer PRA-3000. Magnetic circular dichroism (MCD) measurements were carried out in a 8 kG magnetic field using a JASCO J-20 circular dichrometer. Triplet state formation was observed for investigated compounds at the experimental set up, whose detailed description can be found in our paper (27). The optical experiments were carried out with a porphyrin concentration of 4.10- - 4.10 mol.l". In NMR investigations (Bruker WM-360) we used higher concentrations ( 5.10" raol.l ) and dried solvents (CDCl, C 2 and toluene-d0). 

Fiqure 5. Absorption (a), fluorescence excitation (b) and fluorescence (c) spectra of compound 12 in EPIP at 77 K. Recording (b) and excitation (c) wavelengths are shown in brackets. Correction of exciting light spectral distribution for spectra c has been done up to 600 nm. 

A fluorescence emission spectrum is generally measured by setting the excitation monochromator, Mi, to the chosen wavelength and scanning the second monochromator, M2, with constant slit width. The fluorescent screen monitor, F-P2, now serves to correct for variations in the intensity of the exciting light caused by fluctuations in lamp output. The emission spectrum so recorded has to be corrected for the spectral sensitivity of the apparatus to give the true emission spectrum. 

Summarizing, it is demonstrated that the developed model correctly reproduces the general trends in various experimentally measured responses, which include cuts of time- and frequency-gated spectra at particular frequencies, peak-shifts of the fluorescence spectra, and integral signals. Moreover, the relative shapes and intensities of the spectral cuts at different frequencies are correctly reproduced. For a more complete and quantitative description of the experimental data, the theoretical model has to be augmented by including additional system and/or solvation modes. 

The measured data was corrected for group velocity dispersion by comparison with white light generated in water under the same configuration, yielding in the end a two dimensional array of the fluorescence intensity as a function of frequency and time. Singular value decomposition and deconvolution of the obtained decays was used to reconstruct the spectral decay which would correspond to delta pulse excitation. S(t) was calculated from this processed data. 

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