Limits...
Fluorescence of dyes in solutions with high absorbance. Inner filter effect correction.

Fonin AV, Sulatskaya AI, Kuznetsova IM, Turoverov KK - PLoS ONE (2014)

Bottom Line: Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules.We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object.It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60).

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural dynamics, Stability and Protein folding, Institute of Cytology Russian Academy of Science, St. Petersburg, Russia.

ABSTRACT
Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.

Show MeSH

Related in: MedlinePlus

The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k′ is chosen so that k′I0 = 1, and consequently  numerically equals to q at . The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4114876&req=5

pone-0103878-g001: The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k′ is chosen so that k′I0 = 1, and consequently numerically equals to q at . The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).

Mentions: In the simplest case (AABS = 0, i = 1), AΣ = AFL, and consequently, Eq. 1 will become the following [2]:(2)It is easy to show that fluorescence intensity of a solution can be presented as a linear function of AFL with a slope of q (Figure 1):(3)Here, W is a correction factor:(4)which tends to 2.303 as .


Fluorescence of dyes in solutions with high absorbance. Inner filter effect correction.

Fonin AV, Sulatskaya AI, Kuznetsova IM, Turoverov KK - PLoS ONE (2014)

The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k′ is chosen so that k′I0 = 1, and consequently  numerically equals to q at . The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114876&req=5

pone-0103878-g001: The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k′ is chosen so that k′I0 = 1, and consequently numerically equals to q at . The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).
Mentions: In the simplest case (AABS = 0, i = 1), AΣ = AFL, and consequently, Eq. 1 will become the following [2]:(2)It is easy to show that fluorescence intensity of a solution can be presented as a linear function of AFL with a slope of q (Figure 1):(3)Here, W is a correction factor:(4)which tends to 2.303 as .

Bottom Line: Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules.We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object.It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60).

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural dynamics, Stability and Protein folding, Institute of Cytology Russian Academy of Science, St. Petersburg, Russia.

ABSTRACT
Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.

Show MeSH
Related in: MedlinePlus