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Worldwide comparability of fluorescence measurements

Within a cooperation between PTB, BAM and two partners from industry, spectral fluorescence standards have been developed, characterised and certified, making it possible to eliminate instrument-specific influences on the fluorescence signal. Thus, fluorescence measurements can now be compared worldwide.

Compact integrating sphere radiator (left) and fluorescence standards with the corrected fluorescence spectra (right)

Fluorescence is a widely used analysis method whose applications range from environmental protection (e.g. pollutant determination) to clinical-chemical analysis (e.g. protein and DNA analysis) and the sensitive detection of chromatographic partition processes (e.g. high-performance thin-film chromatography). Fluorescence is therefore measured by means of numerous and various instrumental systems. In order to enable a mutual comparison of the results, it is necessary to detect and correct device-specific influences on the fluorescence signal. A cooperation programme between PTB, the Federal Institute of Materials Research and Testing (BAM) and the companies Gigahertz-Optik GmbH and Sigma-Aldrich GmbH, sponsored by the Federal Ministry of Economics and Technology, has achieved exactly these goals by means of a radiometric traceability chain.

The traceability chain comprises three essential elements: first, a compact, homogeneously radiating integrating sphere source. Its spectral radiance was (with virtually Lambertian emitter characteristics) reduced by several orders of magnitude compared to a tungsten strip lamp, so that its radiation properties are far better adapted to a fluorescent sample than common radiance standards.

The second element is a reference fluorometer with minimal optical imaging errors which — traced back to the radiance standard — allows the certification of fluorescence spectra with sufficiently small radiometric measurement uncertainties.

The third element of the traceability chain is the calibration set "Spectral fluorescence standards BAM-F001 — BAM-F005" whose corrected emission spectra were certified by the BAM in January 2006. These chemical transfer standards allow the user to detect and correct for the relative spectral sensitivity of the emission channel of fluorescence measuring systems under application-relevant conditions in an easy, fast and traceable way. This was confirmed by the national metrology institutes that are active in the field of fluorometry (NIST, NRC, NPL, PTB and BAM) by an international comparison of corrected fluorescence spectra, during which the new calibration set "Spectral fluorescence standards" was used as a transfer standard.