Inner filter corrections

Other corrections, besides those for static interactions, are important for certain quenchers. For example, acrylamide quenching is often used to help determine the relative solvent accessibility of aromatic residue side chains. In addition to a correction for static quenching,(60,66) acrylamide quenching data for tyrosine residues require both primary and secondary inner filter corrections since acrylamide absorbs both 280- and 305-nm light.(67)... 

Fluorescence Data Analysis and Inner Filter Corrections. Figure 2 shows the fluorescence intensity for Fobs versus the concentration of the DOC in ppm for the four san )les. The Fobs that increasing DOC concentrations leads to a decrease... 

The first and second ctors correspond to the primary inner filter and secondary inner filter correction fectors, respectively. For Ax and Ay it was decided to use a value of 0.12 cm and have Xj and y, equal to 0.3 cm. 

The method of fluorescence quenching has been lied to DOC isolated from a stream, wetlands marsh, as well as coniferous and deciduous sites in a local watershed. Uncertainties in the inner filter correction parameters and instability of an aqueous pyrene stock solution introduced some variability into the final binding afiSnity values. The coniferous and deciduous sites had the largest binding afSnities and the greatest amount of DOC per unit volume. The stream and marsh samples had the lower binding... 

Ohno, T., Fluorescence inner-filtering correction for determining the humification index of dissolved organic matter. Environ. Sci. TechnoL, 36, 742, 2000. 

Attention should be paid to possible problems in the measurement of fluorescence quantum yields (some of which are discussed Section 6.1.5) inner filter effects, possible wavelength effects on Op, refractive index corrections, polarization effects, temperature effects, impurity effects, photochemical instability and Raman scattering. 

Inner filter effects due to the presence of other substances When the solution contains other chromophores that absorb light in the same wavelength range as the fluorescent compound under study, the chromophores act as filters at the excitation wavelength and the fluorescence intensity must be multiplied by a correction factor. If the chromophores do not interact with the fluorescent compound, the correction factor is simply the fraction of light absorbed by the compound at the chosen excitation wavelength, so that the corrected fluorescent intensity is given by ... 

All these inner filters effects are difficult to correct and it is advisory to work as much as possible with dilute solutions. 

The factor A/Ap arises from the contribution of the acceptor moiety to the overall absorption at the excitation wavelength 7p. Attention should be paid to the inner filter effect caused by absorption of the acceptor at the emission wavelength of the donor. Some correction may be necessary. 

B. Fanget, O. Devos, and M. Draye, Correction of Inner Filter Effect in Mirror Coating Cells for Trace Level Fluorescence Measurements, Anal. Chem. 2003, 75, 2790. 

Correct the apparent fluorescence intensities for the inner filter effect. 

A protein solution will absorb a certain proportion of the radiation before it reaches the volume of solution whose fluorescence is viewed by the optics of the instrument (see Fig. B3.6.5). For a solution of absorbance 0.1, the intensity at the center of the cell is about 10% less than that of the incident radiation, with a corresponding decrease in the observed fluorescence. Similarly, the fluorescence intensity may be reduced by absorption in the exit path. This is known as the inner filter effect. In practice, it means that the observed fluorescence intensity is directly proportional to protein concentration only at low concentrations, when absorbance is <0.1 at A,e. The apparent fluorescence intensity (Fapp) can be corrected by the following approximation ... 

The fluorescence intensityfor the inner filter effect canbe corrected using Equation (7.15) (Lakowicz, 1999 Albani, 2004) ... 

In general, when one wants to determine if global and/or local structural modifications have occurred within a protein, circular dichroism experiments are performed. Also, one can record the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from one state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

Finally, one should remember that the standard and the molecule to be analyzed should be studied under the same conditions of temperature and solvent viscosity. Also, it is always better to work at low optical densities in order to avoid corrections for the inner filter effect. 

What will happen if you correct the fluorescence intensities of emission and excitation spectra for the inner filter effect ... 

Corrections of the fluorescence excitation and emission spectra for the optical densities (inner filter effect) yield an increase in the intensities without inducing any shift in any of the maxima. This is true for all recorded spectra. At the optical densities we are suggesting, you may find that these corrections are not significant and even not necessary for all fluorophores but ethidium bromide. Its intensity increases without any modification in the peak position. 

The fluorescence estimation may be impaired by the inner filter effect, but this may be made practically negligible by lowering the sample concentration. A maximal total concentration of 25 pM tryptophan is correct, since about 98% of the incident light is then available at the center of a 4-mm cell. 

Step 5 Students will analyze the emission spectra obtained in step 4 with the Burstein equation. Correct the fluorescence spectrum for the inner filter effect before doing the analysis with the Burstein-Emelyanenko method. 

Measure the fluorescence intensity at the peak, correct it for dilution, then for the inner filter effect, and plot the corrected fluorescence intensity as a function of added DNA concentration. Use the data to determine the number of binding sites and the association constant of the complex. 

Plot the fluorescence emission spectra from 290 to 500 nm (Xex = 280 nm) of 1.5 pM BSA solutions prepared at pH 3 and 7. Then, to each cuvette, add 15 pM TNS and record the protein emission spectra at the two pHs. Correct the fluorescence intensity for the inner filter effect, and then plot the corrected emission spectra. What do you notice Are the spectra obtained at the two pHs identical What are the values of the emission peaks you observe Explain the results you obtained. 

Measure the fluorescence intensities at 335 nm at the different TNS concentrations, and correct them first for the dilution then for the inner filter effect. Finally, plot the fluorescence intensity at 335 nm as a function ofTNS concentration. What do you observe ... 

Figure 15.1 Fluorescence emission of BSA recorded in the absence (a) and presence of TNS (b) at pH 3. Spectra are corrected for the inner filter effect. Xex = 280 nm.

The data shown before are from spectra not corrected for the inner filter effect. The optical density measured at the excitation wavelength (280 nm) and at two emission wavelengths, 335 for experiment 1 and 355 for experiment 2, gives the following values ... 

Correction for the inner filter effect The optical densities are obtained with a spectrophotometer using a path length equal to 1 cm. However, when fluorescence experiments were performed, the optical path length upon excitation was equal to 0.4 cm. Thus, the real optical density at the excitation wavelength is equal to that measured on the spectrophotometer divided by 2.5. ... 

One can notice that after correction for the inner filter effect, one can still observe a decrease in the fluorescence intensities of the Trp residues. This decrease is a result of the TNS binding to the flavodehydrogenase. 

One can see that after correction for the dilution and the inner filter effect, the measured intensities in the presence of TNS reach that measured for the protein alone. This indicates that binding of TNS to the flavodehydrogenase does not occur, and the protein has lost its specific binding site for the TNS. 

With HTRF, the only possible interferences that are not easily corrected are due to the inner filter effect at the acceptor emission wavelength. However, only very few compounds in the hbraries absorb highly in the near infrared region. Possible cryptate quenching is even corrected to a certain extent by the signal ratio, while only acceptor quenching is not taken into account, similar to the situation with the other technologies. 

Fluorescence quantum efficiencies of several solid materials have been measured by photoacoustic spectroscopy." The photophysics of quantities for some common fluorescence standards have been made with some accuracy the influence of refractive index corrections on yield and lifetimes are discussed, 9,10-diphenylanthracene, quinine bisulphate, and 2-aminopyridine being the selected examples. Correction for inner filter effects in fluorescence spectroscopy have been proposed. ... 

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