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fulltext6 - Standardization of Fluorescence Measurements...

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Standardization of Fluorescence Measurements Criteria for the Choice of Suitable Standards and Approaches to Fit-for-Purpose Calibration Tools U TE R ESCH -G ENGER , K ATRIN H OFFMANN , AND A NGELIKA H OFFMANN Federal Institute for Materials Research and Testing (BAM), Div. I.5, D-12489 Berlin, Germany This report summarizes problems associated with the comparability of measurements of photolu- minescence and procedures for the characterization of relevant instruments, focusing on physical and chemical fluorescence standards. To provide recommendations on selecting and using such standards, we derive general and scope-specific requirements and quality criteria for suitable devices and materials and briefly address metrological requirements linked to the realization of comparable measurements. Special emphasis is dedicated to liquid and solid chromophore-based fluorescence standards developed or currently tested by us. Key words: fluorescence standard; fluorescence spectroscopy; spectral correction; day-to-day in- tensity standard; calibration, metrology; fluorescence microscopy Introduction Photoluminescence techniques, which yield analyte- specific quantities, such as emission and excitation spectra, luminescence quantum yields, luminescence lifetimes, and emission anisotropies, are among the most widely used tools in the material and the life sci- ences. These techniques offer comparative ease of use; unique sensitivity; noninvasive character; potential for combining spectrally, temporally, and spatially resolved measurements; and suitability for multiplexing and re- mote sensing. 1 , 2 However, all photoluminescence tech- niques suffer from a series of drawbacks. First, mea- sured signals include unwanted contributions from instrument-dependent effects that are wavelength-, polarization-, and (because of aging of optical and electro-optical instrument components) time depen- dent. Second, the spectroscopic properties of most chromophores (e.g., absorption and emission spectra, molar absorption coefficient, luminescence quantum yield, luminescence lifetime, and luminescence po- larization or anisotropy) are influenced by their mi- croenvironment (in terms of temperature, viscosity, solvation, polarity, proticity, pH, ionic strength, pres- Address for correspondence: Dr. Ute Resch-Genger, Federal Institute for Materials Research and Testing (BAM), I.5 Bioanalytics, Richard- Willstaetter-Str. 11, 12489 Berlin-Adlershof, Germany. Voice: 030-8104- 1134; fax: 030-8104-1157. [email protected] ence of quenchers, and attachment to bio- or macro- molecules). Finally, there are significant challenges to measuring absolute luminescence intensities. 3 7 These luminescence-inherent drawbacks hamper the com- parability of luminescence data between instruments and can render quantification from measurements of relative fluorescence intensities erroneous. These limi- tations can be overcome with simple, robust, and flexi- ble standards that enable a straightforward instrument characterization under application-relevant conditions and can be used for different fluorescence techniques.
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