Recommendations for Fluorescence Instrument Qualification: The

Feb 5, 2010 - The new ASTM Standard Guide for Fluorescence—Instrument Calibration ... uncertainties, related references, and general instructions fo...
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Anal. Chem. 2010, 82, 2129–2133

Recommendations for Fluorescence Instrument Qualification: The New ASTM Standard Guide Paul C. DeRose*,† and Ute Resch-Genger‡ National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, Maryland 20899-8312, and BAM Federal Institute for Materials Research and Testing, Division I.5, Richard-Willstaetter-Str. 11, D-12489 Berlin, Germany Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide’s main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-today performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements. Recent developments in quantitative fluorescence-based assays in clinical, pharmaceutical, biotechnological, and other areas, in conjunction with global trends to harmonize measurements, traceability, and accreditation,1,2 have spurred the demand for fluorescence standards and related standardization documents. The latter include carefully evaluated standard operating procedures, guidelines, and recommendations for instrument characterization and performance verification. Fluorescence standards include physical standards, e.g., a calibrated light source, and chemical standards, such as solid or liquid reference materials. Suitable examples should be robust, easy-to-use, readily available and, when appropriate, given with values that are SI-traceable, * To whom correspondence should be addressed. Tel.: 301-975-4572. Fax: 301-977-0587. E-mail: [email protected]. † National Institute of Standards and Technology. ‡ BAM Federal Institute for Materials Research and Testing. (1) Saunders, G.; Parkes, H. Analytical Molecular Biology: Quality and Validation; RSC: Cambridge, 1999. (2) ISO/IEC 17025, General Requirements for the Competence of Testing and Calibration Laboratories, 2nd ed.; International Organization for Standardization: Geneva, 2005. 10.1021/ac902507p  2010 American Chemical Society Published on Web 02/05/2010

that is, traceable to the Syste´me Internationale (SI), which is the internationally recognized system of units (see traceability in the Termology section). This combination of material and written standards will provide the prerequisites for the eventual and desired standardization of fluorescence measurements. These demands have been met, in part, by new certified reference materials, recently released by National Metrology Institutes,3-9 i.e., the National Institute of Standards and Technology (NIST) and the Federal Institute for Materials Research and Testing (BAM), intended for obtaining spectral correction of detection systems, day-to-day performance verification of instruments, and calibration of signal to fluorophore/analyte concentration. The increasing importance of fluorescence measurements has also encouraged scientific organizations like the International Union of Pure and Applied Chemistry (IUPAC; fluorometry task force group) and standards organizations such as ASTM International (formerly the American Society for Testing of Materials) to respond to this increase in demand with guidelines and recommendations documents for fluorescence.10-17 (3) Certificate of Analysis, Standard Reference Material 2940: Relative Intensity Correction Standard for Fluorescence Spectroscopy: Orange Emission; National Institute of Standards and Technology, Gaithersburg: MD, 2007; https:// www-s.nist.gov/srmors/view_detail.cfm?srm)2940. (4) Certificate of Analysis, Standard Reference Material 2941: Relative Intensity Correction Standard for Fluorescence Spectroscopy: Green Emission; National Institute of Standards and Technology: Gaithersburg, MD, 2007; https:// www-s.nist.gov/srmors/view_detail.cfm?srm)2941. (5) Certificate of Analysis, Standard Reference Material 2942: Relative Intensity Correction Standard for Fluorescence Spectroscopy: Ultraviolet Emission; National Institute of Standards and Technology: Gaithersburg, MD, 2009; https://www-s.nist.gov/srmors/view_detail.cfm?srm)2942. (6) Certificate of Analysis, Standard Reference Material 2943: Relative Intensity Correction Standard for Fluorescence Spectroscopy: Blue Emission; National Institute of Standards and Technology: Gaithersburg, MD, 2009; https:// www-s.nist.gov/srmors/view_detail.cfm?srm)2943. (7) Certificate of Analysis, Certified Reference Materials BAM-F001, -F002a, -F003-F005, Calibration Kit, Spectral Fluorescence Standards; Federal Institute for Materials Research and Testing: Berlin, 2009. These are available from BAM or from Sigma-Aldrich. (8) Certificate of Analysis, Certified Reference Materials BAM-F001-BAM-F005, Calibration Kit, Spectral Fluorescence Standards; Federal Institute for Materials Research and Testing: Berlin, 2006. (9) Certificate of Analysis, Standard Reference Material 1932: Fluorescein Solution, National Institute of Standards and Technology: Gaithersburg, MD, 2003; https://www-s.nist.gov/srmors/view_detail.cfm?srm)1932. (10) DeRose, P. C. Standard Guide to FluorescencesInstrument Calibration and Validation, NISTIR 7458; National Institute of Standards and Technology: Gaithersburg, MD, 2007(submitted to ASTM for revision and publication). (11) Resch-Genger, U.; DeRose, P. C. Fluorescence Standards: Classification, Terminology and Recommendations On Their Selection, Use and Production. Pure Appl. Chem., in press. (12) IUPAC Project no. 2004 021 1 300.

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ASTM International has provided standard test methods for fluorescence spectrometry since 1972 under the jurisdiction of subcommittee E13.01 Ultraviolet, Visible and Luminescence Spectroscopy. The three pre-existing ASTM standards presently available are tests for wavelength accuracy and spectral resolution (E 388),18 detection system linearity (E 578),19 and limit of detection (E 579).20-22 The new ASTM Standard Guide for FluorescencesInstrument Calibration and Qualification,23 reviewed here, discusses physical and chemical fluorescence standards, and instrument and analyte characterization procedures along with their purpose, uncertainties, related references, and general instructions for use. The guide focuses on the most relevant instrument properties to be qualified and also introduces procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes. In addition, a General Guidelines section alerts nonexperts to precautions that can reduce the risk of errors and measurement uncertainties. Cuvette quality, solvent selection, common contaminants, and other sample and instrument related best practices are discussed. A more in-depth summary of some of the core sections of the guide is given in what follows. TERMINOLOGY More than 50 terms that are commonly used in fluorometry and instrument qualification are given in the guide. Here is a sampling of those terms that are also used in this review. Calibration. A set of procedures that establishes the relationship between quantities measured on an instrument and the corresponding values realized by standards. (13) DeRose, P. C.; Resch-Genger, U.; Wang, L.; Gaigalas, A. K.; Kramer, G. W.; Panne, U. In Standardization in Fluorometry: State-of-the Art and Future Challenges; Springer Series on Fluorescence; Resch-Genger, U., Ed.; Springer-Verlag GmbH: Berlin Heidelberg, 2008; Vol. 5, pp 33-62. (14) DeRose, P. C. Recommendations and Guidelines for Standardization of Fluorescence Spectroscopy, NISTIR 7457; National Institute of Standards and Technology: Gaithersburg, MD, 2007. (15) Marti, G. E.; Vogt, R. F.; Gaigalas, A. K.; Hixson, C. S.; Hoffman, R. A.; Lenkei, R.; Magruder, L. E.; Purvis, N. B.; Schwartz, A.; Shapiro, H. M.; Waggoner A. Fluorescence Calibration and Quantitative Measurements of Fluorescence Intensity, Approved Guideline, NCCLS, I/LA24-A, 2004; Vol. 24 No. 26. (16) Resch-Genger, U.; Hoffmann, K.; Pfeifer, D. In Reviews in Fluorescence 2007, Geddes, C. D., Ed.; Springer Science Businesss Media, Inc.: New York, 2009; pp 1-32. (17) Resch-Genger, U.; Hoffmann, K.; Hoffmann, A. Ann. N.Y. Acad. Sci. 2008, 1130, 35–43. (18) ASTM E 388-04, Standard Test Method for Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers. In Annual book of ASTM standards; 2004; Vol 03.06 (original version 1972). (19) ASTM E 578-07, Standard Test Method for Linearity of Fluorescence Measuring Systems. In Annual book of ASTM standards; ASTM International: West Conshohocken, PA, 2007; Vol 03.06 (original version 1983). (20) ASTM E 579-04, Standard Test Method for Limit of Detection of Fluorescence of Quinine Sulfate. In Annual book of ASTM standards; ASTM International: West Conshohocken, PA, 2004; Vol 03.06 (original version 1984). (21) Certificate of analysis, Standard Reference Material 936, quinine sulfate dihydrate; National Institute of Standards and Technology: Gaithersburg, MD, 1979. (22) Certificate of analysis, Standard Reference Material 936a, quinine sulfate dihydrate; National Institute of Standards and Technology: Gaithersburg, MD, 1994; https://www-s.nist.gov/srmors/view_detail.cfm?srm)936a. (23) ASTM E 2719, Standard Guide for FluorescencesInstrument Calibration and Validation. In Annual book of ASTM standards; ASTM International: West Conshohocken, PA, 2010; Vol 03.06.

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Certified Value. The value for which the certifying body has the highest confidence in its accuracy in that all known or suspected sources of bias have been investigated or accounted for by the certifying body.24 The certifying body, typically a national metrology institute or a commercial producer of reference materials, is the organization that measures and reports the certified value and the bias related with that value. Limit of Detection. An estimate of the lowest concentration of an analyte that can be measured with a given technique, often taken to be the analyte concentration with a measured signal-tonoise ratio of three. Qualification. The process producing evidence that an instrument consistently yields measurements meeting required specifications and quality characteristics. Traceability. Linking of the value and uncertainty of a measurement to the highest reference standard or value through an unbroken chain of comparisons. In this definition, highest refers to the reference standard whose value and uncertainty are not dependent on those of any other reference standards, and unbroken chain of comparisons refers to the requirement that any intermediate reference standards used to trace the measurement to the highest reference standard must have their values and uncertainties linked to the measurement as well.25,26 FLUORESCENCE INSTRUMENT QUALIFICATION Reliable and comparable fluorescence measurements require qualification of some or all of the following instrument properties, depending on the type of instrument and application: wavelength and spectral slit width accuracy, linearity of the detection system, spectral responsivity of the emission detection system (spectral correction of the detection system responsivity or emission correction), spectral irradiance reaching the sample (spectral correction of the excitation beam intensity), sensitivity (limit of detection for an analyte), and day-to-day performance verification. For each instrument property, different methods and suitable standards are presented, and the advantages and disadvantages of each method are discussed to enable users of fluorescence techniques to determine which method is the best choice for their needs and the easiest for them to implement. The diverse entries are at a variety of quality and expertise levels with the intent of satisfying a wide variety of users. Wavelength Accuracy. There are a variety of commercially available samples and lamps that can be used to determine the wavelength accuracy of either emission detection systems or excitation wavelength selectors, or both, within the UV/vis/NIR spectral region. Eight different methods are presented, using atomic pen lamps,18 rare earth doped crystals27 and glasses,28 (24) May, W.; Parris, R.; Beck, C.; Fassett, J.; Greenberg, R.; Guenther, F.; Kramer, G.; Wise, S.; Gills, T.; Colbert, J.; Gettings, R.; MacDonald, B. Definitions of Terms and Modes Used at NIST for Value-assignment of Reference Materials for Chemical Measurements; NIST Special Publication 260-136; U.S. Government Printing Office: Washington, DC, 2000. (25) International vocabulary of basic and general terms in metrology (VIM), 3rd ed.; 2004. (26) International vocabulary of basic and general terms in metrology (VIM), 2nd ed.; Beuth Verlag: Berlin, 1994. (27) Lifshits, I. T.; Meilman, M. L. Sov. J. Opt. Technol. 1988, 55, 487–489. (28) Velapoldi, R. A.; Epstein, M. S. In ACS Symposium Series 383, Luminescence Applications in Biological, Chemical, Environmental and Hydrological Sciences; Goldberg, M. C., Ed.; American Chemical Society: Washington, DC, 1989; pp 98-126.

Table 1. Summary of Methods for Determining Wavelength Accuracy sample

λ region

uncertainty

limitations

UV-NIR (EM) ±0.1 nm or better alignment 470-760 nm (EM) ±0.1 nm 255-480 nm (EX) Eu glass 570-700 nm (EM) ±0.2 nm 360-540 nm (EX) anthracene in PMMA 380-450 nm (EM) ±0.2 nm limited λ range 310-380 nm (EX) Ho2O3 + diff-use reflector 330-800 nm (EM or EX) ±0.4 nm need blank Xe source 400-500 nm (EX) ±0.2 nm limited λ range, calibration Xe source + diffuse reflector UV-NIR ±0.2 nm monochromator must be calibrated Water raman UV-blue ±0.2 nm monochromator must be calibrated pen lamp Dy-YAG crystal

established values

refs

Y Y

18 27

N

28

N Y N Y N

29-31 32 32 33, 34

Ho2O3 samples,29-31 Xe source lamps,32 or just water33,34 (see Table 1). Samples with multiple, narrow, well-defined peaks covering a very broad wavelength region are the best candidates for reference materials of this type. For some samples, e.g., atomic lamps, reference values of wavelengths have been certified by national metrology institutes. For others, these values have been established in the literature, e.g., a Dy-YAG crystal. Then, there are some samples whose values have not been established, even though they are sold as “wavelength standards”, e.g., anthracene in PMMA. Even samples with unestablished values can be used for wavelength accuracy determinations, if their values are predetermined by the user, using one of the established methods. Linearity of the Detection System. The responsivity of detection systems is not linear with signal intensity at all intensity levels. High intensity levels are most problematic because the detector may become saturated, causing potentially large deviations from linear behavior. This represents one of the largest sources of uncertainty for fluorescence measurements. Linearity, as well as spectral correction (see next section), is particularly important for quantification of analyte concentration from measured fluorescence intensities and for applications where intensity ratios or spectral shapes need to be measured with accuracy. Presented methods include the use of a double aperture,35,36 optical filters or polarizers,32,37,38 and reference samples with a range of fluorophore concentrations.19 The last of these is generally the easiest to implement. Spectral Correction. Correction of detection system responsivity as a function of emission wavelength enables true instru(29) Certificate of Analysis, Standard Reference Material 2065 Ultraviolet-VisibleNear-Infrared Transmission Wavelength Standard; National Institute of Standards and Technology: Gaithersburg, MD, 2002; https://www-s.nist. gov/srmors/view_detail.cfm?srm)2065. (30) Certificate of Analysis, Standard Reference Material 2034 Holmium Oxide Solution; National Institute of Standards and Technology: Gaithersburg, MD, 1985; https://www-s.nist.gov/srmors/view_detail.cfm?srm)2034. (31) Paladini, A. A.; Erijman, L. J. Biochem. Biophys. Methods 1988, 17, 61–66. (32) DeRose, P. C.; Early, E. A.; Kramer, G. W. Rev. Sci. Instrum. 2007, 78, 033107. (33) Technical Note: The Measurement of Sensitivity in Fluorescence Spectroscopy; Photon Technology International, 2005; http://www.pti-nj.com/products/ Steady-State-Spectrofluorometer/TechNotes/MeasurementSensitivity.pdf. (34) Sensivity of the Fluorolog and FluoroMax Spectrofluorometers; HORIBA Jobin Yvon, 2007; http://www.horiba.com/fileadmin/uploads/Scientific/ Documents/ Fluorescence/Raman_sensitivity_FL-13.pdf. (35) Mielenz, K. D.; Eckerle, K. L. Appl. Opt. 1972, 11, 2294–2303. (36) Zwinkels, J. C.; Gignac, D. S. Appl. Opt. 1991, 30, 1678–1687. (37) Hollandt, J.; Taubert, R. D.; Seidel, J.; Resch-Genger, U.; Gugg-Helminger, A.; Pfeifer, D.; Monte, C.; Pilz, W. J. Fluoresc. 2005, 15, 301–313. (38) Resch-Genger, U.; Pfeifer, D.; Monte, C.; Pilz, W.; Hoffmann, A.; Spieles, M.; Rurack, K.; Hollandt, J.; Taubert, D.; Scho¨nenberger, B.; Nording, P. J. Fluoresc. 2005, 15, 315–336.

Figure 1. Comparison of a spectrally corrected ( · · · · ) and an uncorrected (s) fluorescence emission spectrum for (a) standard reference material (SRM) 2941, green emission standard, taken on a fluorescence spectrometer with a monochromator/photomultiplier tube based detection system (Horiba Jobin Yvon) and 427 nm lamp excitation and (b) SRM 2940, orange emission standard, taken on a fluorescence spectrometer with a grating/CCD based detection system (Horiba Jobin Yvon) and 404 nm laser excitation.39

ment-independent emission spectra to be obtained. Typical deviations of an uncorrected spectrum from its true (corrected) values are shown in Figure 1,39 using a calibrated light source (CS)-based spectral correction method, thereby underlining the importance of spectral correction. An uncorrected spectrum can have significantly different peak ratios (see Figure 1a), shape, peak (39) Gaigalas, A. K.; Wang, L.; He, H.-J.; DeRose, P. C. J. Res. NIST 2009, 114, 215–228.

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Table 2. Summary of Methods for Determining the Spectral Responsivity of the Detection System (Emission Correction) sample

λ region

uncertainty

limitations

certified values

refs

calibrated light source calibrated detector + calibrated reflector certified reference materials

UV-NIR UV-NIR UV-NIR