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Relative and absolute determination of fluorescence quantum yields of transparent samples

Nature Protocols volume 8, pages 1535–1550 (2013)Cite this article

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Abstract

Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ∼350–950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5–10% can be achieved within 2 h.

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Figure 1: Absorption (top) and emission (bottom) spectra of the recommended quantum yield standards.
Figure 2
Figure 3: Scheme of a fluorescence spectrometer (configuration A) and an integrating sphere setup (configuration B).
Figure 4: Absorption spectra of the sample (Ax) and the standard (Ast).
Figure 5: Example emission spectra of the sample (Ic,x) and the standard (Ic,st).
Figure 6: Example of the signals of an integrating sphere measurement for the sample and the blank.
Figure 7: The effect of spectral correction on the shape and relative intensities of the emission spectra of two dyes.
Figure 8: Visualization of the effect of a scattering background on a dye's absorption spectrum.
Figure 9: Distortion of the emission spectrum as a result of fluorescence reabsorption.
Figure 10: Typical changes in the absorption spectrum caused by dye aggregation (H-type aggregates).
Figure 11: Concentration dependence of the fluorescence quantum yield of two CdTe quantum dots differing in particle size.

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Acknowledgements

We gratefully acknowledge financial support from the Federal Ministry of Economics and Technology (BMWI; program 'Messen, Normen, Prüfen, und Qualitätssicherung' (MNPQ), projects BMWI-22/06, BMWI-17/07 and BMWI-13/09) and from the European Union (European Metrology Research Programme (EMRP), grant NanaChOp, NEW03).

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  1. Christian Würth and Markus Grabolle: These authors contributed equally to this work.

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  1. Bundesanstalt für Materialforschung und–prüfung (BAM), Berlin, Germany

    Christian Würth, Markus Grabolle, Jutta Pauli, Monika Spieles & Ute Resch-Genger

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Contributions

All authors contributed to the concept of the manuscript and to the Experimental design and MATERIALS sections. The 'General remarks' (INTRODUCTION) and section were written by C.W. and M.G. with aid from U.R.-G. The procedure for relative quantum yield measurements was developed and written by M.G., J.P. and C.W. The procedure for absolute quantum yield measurements was developed, simplified and written by C.W. M.S. contributed technically, with, e.g., new measurements of dye purity. U.R.-G. initiated this research, wrote the introduction and gave an overview of different techniques for the determination of photoluminescence quantum yields including topics such as limitations, method development and fluorescence standards.

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Supplementary information

Supplementary Methods

Determination of the spectral responsivity of fluorescence instruments (PDF 1715 kb)

Supplementary Data

Chemical data for the standards used in the protocol (PDF 573 kb)

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Würth, C., Grabolle, M., Pauli, J. et al. Relative and absolute determination of fluorescence quantum yields of transparent samples. Nat Protoc 8, 1535–1550 (2013). https://doi.org/10.1038/nprot.2013.087

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