INNER FILTER EFFECT FLUORESCENCE

MacDonald, B. C. Lvin, S. J., and Patterson, H. H. Correction of Fluorescence Inner Filter Effects and the Partitioning of Pyrene to Dissolved Organic Carbon Submitted to Analy. Chimica Acta, 1996. 

Kao, S., Asanov, A.N., Oldham, P.B. (1998) A Comparison of Fluorescence Inner Filter Effects for Different Cell Configurations. Instrumentation Science Technology, Vol. 26, No. 4, pp. 375-387, ISSN 1073-9149. 

Moreover, when the concentration of fluorescent compound is high, inner filter effects reduce the fluorescence intensity depending on the observation conditions (see Chapter 6). In particular, the photons emitted at wavelengths corresponding to the overlap between the absorption and emission spectra can be reabsorbed (radiative transfer). Consequently, when fluorometry is used for a quantitative evaluation of the concentration of a species, it should be kept in mind that the fluorescence intensity is proportional to the concentration only for diluted solutions. 

Radiative transfer results in a decrease of the donor fluorescence intensity in the region of spectral overlap. Such a distortion of the fluorescence spectrum is called the inner filter effect (see Chapter 6). 

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. 

Pitfalls in steady-state fluorescence measurements inner filter effects and polarization effects... 

Emission inner filter effect (self-absorption) The fluorescence photons emitted in the region overlapping the absorption spectrum can be absorbed (radiative energy trans-... 

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 ... 

Time-resolved emission of the donor or acceptor fluorescence provides direct information on the transfer rate, without the difficulties that may result from inner filter effects. 

S. P. Srinivas and R. Mutharasan, Inner filter effects and their interferences in the interpretation of culture fluorescence, Biotech, and Bioeng. 30, 769-774(1987). 

Inner-filter effects. The absorption of the fluorescence excitation and emission by the specimen is referred to as the "inner-filter" effect this effect has been treated in the literature (24-27). The inner-filter effect reduces the signal levels and distorts the emission spectrum and the intensity-concentration relationship. The effect is more pronounced in right-angle fluorescence measurements (27) than in the "front face" configuration in which the fluorescence is viewed from the same side as the excitation beam. 

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 ... 

Figure B3.6.5 The inner filter effect. A cuvette (10 x 10-mm) is represented in plan view, with the collimated incident beam from the monochromator having intensity /0. As a result of absorption by the protein solution, the intensity of the beam through the cuvette will decrease steadily, emerging with intensity /. The values are illustrated for a solution having an absorbance at the excitation wavelength of 0.1. The optics of the fluorescence detector are focused so that only fluorescence originating from the volume depicted by the heavily shaded square is seen by the photomultiplier. Thus the observed normalized fluorescence intensity will be less than that expected from the protein at infinite dilution. The fluorescence passes through the protein solution on its way to the detector and will be further decreased in intensity if the solution absorbs at the wavelengths of the emitted radiation.

However, there is a probability that the sample absorbs the wavelength of emitted light, thus inducing fluorescence reduction (inner filter effect). In this case, fluorescence, as a function of concentration, presents another behavior. The effective fluo-... 

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. 

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. 

---