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

PLoS One. 2014 Jul 29;9(7):e103878. doi: 10.1371/journal.pone.0103878. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Filtration / instrumentation
  • Fluorescent Dyes / chemistry*
  • Quantum Theory
  • Solutions / chemistry*
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods*

Substances

  • Fluorescent Dyes
  • Solutions

Grants and funding

This work was supported by the Program “Molecular and Cell Biology” of the Russian Academy of Sciences, Russian Foundation of Basic Research, grant 14-04-90024_Bel (K.K.T.), 12-04-01651(K.K.T.), 12-04-31708 (A.V.F.), 13-04-01842 (I.M.K.), 13-04-02068 (A.I.S.) by Ministry of Education and Science of the Russian Federation, Agreements 14.132.21.1311 (A.I.S.) and 8830 (K.K.T.) and by RF President fellowships SP-2390.2012.4 (A.V.F.) and SP-776.2012.4 (A.I.S.). Fluorescence measurements by Fluorolog-3 (Horiba, Japan) were carried out in the Resource Center for Optical and Laser Materials Research of St. Petersburg State University, Russia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.