Recommendations for Fluorescence InstrumentQualification: 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 BAMFederal Institute for Materials Research and Testing, Division I.5, Richard-Willstaetter-Str. 11,D-12489 Berlin, Germany

Aimed at improving quality assurance and quantitationfor modern fluorescence techniques, ASTM International(ASTM) is about to release a Standard Guide for Fluores-cence, reviewed here. The guide’s main focus is on steadystate fluorometry, for which available standards andinstrument characterization procedures are discussedalong with their purpose, suitability, and general instruc-tions for use. These include the most relevant instrumentproperties needing qualification, such as linearity andspectral responsivity of the detection system, spectralirradiance reaching the sample, wavelength accuracy,sensitivity or limit of detection for an analyte, and day-to-day performance verification. With proper considerationof method-inherent requirements and limitations, manyof these procedures and standards can be adapted toother fluorescence techniques. In addition, proceduresfor the determination of other relevant fluorometric quan-tities including fluorescence quantum yields and fluores-cence lifetimes are briefly introduced. The guide is a clearand concise reference geared for users of fluorescenceinstrumentation at all levels of experience and is intendedto aid in the ongoing standardization of fluorescencemeasurements.

Recent developments in quantitative fluorescence-based assaysin clinical, pharmaceutical, biotechnological, and other areas, inconjunction with global trends to harmonize measurements,traceability, and accreditation,1,2 have spurred the demand forfluorescence standards and related standardization documents.The latter include carefully evaluated standard operating proce-dures, guidelines, and recommendations for instrument charac-terization and performance verification. Fluorescence standardsinclude physical standards, e.g., a calibrated light source, andchemical standards, such as solid or liquid reference materials.Suitable examples should be robust, easy-to-use, readily availableand, when appropriate, given with values that are SI-traceable,

that is, traceable to the Systeme Internationale (SI), which is theinternationally recognized system of units (see traceability in theTermology section). This combination of material and writtenstandards will provide the prerequisites for the eventual anddesired standardization of fluorescence measurements. Thesedemands have been met, in part, by new certified referencematerials, 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 detectionsystems, day-to-day performance verification of instruments, andcalibration of signal to fluorophore/analyte concentration. Theincreasing importance of fluorescence measurements has alsoencouraged scientific organizations like the International Unionof Pure and Applied Chemistry (IUPAC; fluorometry task forcegroup) and standards organizations such as ASTM International(formerly the American Society for Testing of Materials) torespond to this increase in demand with guidelines and recom-mendations documents for fluorescence.10-17

* To whom correspondence should be addressed. Tel.: 301-975-4572. Fax:301-977-0587. E-mail: paul.derose@nist.gov.

† National Institute of Standards and Technology.‡ BAM Federal Institute for Materials Research and Testing.

(1) Saunders, G.; Parkes, H. Analytical Molecular Biology: Quality and Valida-tion; RSC: Cambridge, 1999.

(2) ISO/IEC 17025, General Requirements for the Competence of Testing andCalibration Laboratories, 2nd ed.; International Organization for Standardiza-tion: Geneva, 2005.

(3) Certificate of Analysis, Standard Reference Material 2940: Relative IntensityCorrection Standard for Fluorescence Spectroscopy: Orange Emission; NationalInstitute 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 IntensityCorrection Standard for Fluorescence Spectroscopy: Green Emission; NationalInstitute 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 IntensityCorrection 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 IntensityCorrection Standard for Fluorescence Spectroscopy: Blue Emission; NationalInstitute 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; FederalInstitute for Materials Research and Testing: Berlin, 2009. These areavailable from BAM or from Sigma-Aldrich.

(8) Certificate of Analysis, Certified Reference Materials BAM-F001-BAM-F005,Calibration Kit, Spectral Fluorescence Standards; Federal Institute forMaterials Research and Testing: Berlin, 2006.

(9) Certificate of Analysis, Standard Reference Material 1932: FluoresceinSolution, 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 andValidation, 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 Produc-tion. Pure Appl. Chem., in press.

(12) IUPAC Project no. 2004 021 1 300.

Anal. Chem. 2010, 82, 2129–2133

10.1021/ac902507p 2010 American Chemical Society 2129Analytical Chemistry, Vol. 82, No. 5, March 1, 2010Published on Web 02/05/2010

ASTM International has provided standard test methods forfluorescence spectrometry since 1972 under the jurisdiction ofsubcommittee E13.01 Ultraviolet, Visible and Luminescence Spec-troscopy. The three pre-existing ASTM standards presentlyavailable are tests for wavelength accuracy and spectral resolution(E 388),18 detection system linearity (E 578),19 and limit ofdetection (E 579).20-22

The new ASTM Standard Guide for FluorescencesInstrumentCalibration and Qualification,23 reviewed here, discusses physicaland chemical fluorescence standards, and instrument and analytecharacterization procedures along with their purpose, uncertain-ties, related references, and general instructions for use. The guidefocuses on the most relevant instrument properties to be qualifiedand also introduces procedures for the determination of otherrelevant fluorometric quantities including fluorescence quantumyields and fluorescence lifetimes. In addition, a General Guidelinessection alerts nonexperts to precautions that can reduce the riskof errors and measurement uncertainties. Cuvette quality, solventselection, common contaminants, and other sample and instru-ment related best practices are discussed. A more in-depthsummary of some of the core sections of the guide is given inwhat follows.

TERMINOLOGYMore than 50 terms that are commonly used in fluorometry

and instrument qualification are given in the guide. Here is asampling of those terms that are also used in this review.

Calibration. A set of procedures that establishes the relation-ship between quantities measured on an instrument and thecorresponding values realized by standards.

Certified Value. The value for which the certifying body hasthe highest confidence in its accuracy in that all known orsuspected sources of bias have been investigated or accountedfor by the certifying body.24 The certifying body, typically anational metrology institute or a commercial producer of referencematerials, is the organization that measures and reports thecertified value and the bias related with that value.

Limit of Detection. An estimate of the lowest concentrationof an analyte that can be measured with a given technique, oftentaken to be the analyte concentration with a measured signal-to-noise ratio of three.

Qualification. The process producing evidence that an instru-ment consistently yields measurements meeting required speci-fications and quality characteristics.

Traceability. Linking of the value and uncertainty of ameasurement to the highest reference standard or value throughan unbroken chain of comparisons. In this definition, highest refersto the reference standard whose value and uncertainty are notdependent on those of any other reference standards, andunbroken chain of comparisons refers to the requirement that anyintermediate reference standards used to trace the measurementto the highest reference standard must have their values anduncertainties linked to the measurement as well.25,26

FLUORESCENCE INSTRUMENT QUALIFICATIONReliable and comparable fluorescence measurements require

qualification of some or all of the following instrument properties,depending on the type of instrument and application: wavelengthand spectral slit width accuracy, linearity of the detection system,spectral responsivity of the emission detection system (spectralcorrection of the detection system responsivity or emissioncorrection), spectral irradiance reaching the sample (spectralcorrection of the excitation beam intensity), sensitivity (limit ofdetection for an analyte), and day-to-day performance verification.For each instrument property, different methods and suitablestandards are presented, and the advantages and disadvantagesof each method are discussed to enable users of fluorescencetechniques to determine which method is the best choice for theirneeds and the easiest for them to implement. The diverse entriesare at a variety of quality and expertise levels with the intent ofsatisfying a wide variety of users.

Wavelength Accuracy. There are a variety of commerciallyavailable samples and lamps that can be used to determine thewavelength accuracy of either emission detection systems orexcitation wavelength selectors, or both, within the UV/vis/NIRspectral region. Eight different methods are presented, usingatomic pen lamps,18 rare earth doped crystals27 and glasses,28

(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 FutureChallenges; 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 ofFluorescence Spectroscopy, NISTIR 7457; National Institute of Standards andTechnology: 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 ofFluorescence 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 andWavelength Accuracy of Fluorescence Spectrometers. In Annual book ofASTM standards; 2004; Vol 03.06 (original version 1972).

(19) ASTM E 578-07, Standard Test Method for Linearity of FluorescenceMeasuring Systems. In Annual book of ASTM standards; ASTM Interna-tional: West Conshohocken, PA, 2007; Vol 03.06 (original version 1983).

(20) ASTM E 579-04, Standard Test Method for Limit of Detection of Fluores-cence of Quinine Sulfate. In Annual book of ASTM standards; ASTMInternational: West Conshohocken, PA, 2004; Vol 03.06 (original version1984).

(21) Certificate of analysis, Standard Reference Material 936, quinine sulfatedihydrate; National Institute of Standards and Technology: Gaithersburg,MD, 1979.

(22) Certificate of analysis, Standard Reference Material 936a, quinine sulfatedihydrate; 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 Calibrationand Validation. In Annual book of ASTM standards; ASTM International:West Conshohocken, PA, 2010; Vol 03.06.

(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 ofReference Materials for Chemical Measurements; NIST Special Publication260-136; U.S. Government Printing Office: Washington, DC, 2000.

(25) International vocabulary of basic and general terms in metrology (VIM), 3rded.; 2004.

(26) International vocabulary of basic and general terms in metrology (VIM), 2nded.; 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 HydrologicalSciences; Goldberg, M. C., Ed.; American Chemical Society: Washington,DC, 1989; pp 98-126.

2130 Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

Ho2O3 samples,29-31 Xe source lamps,32 or just water33,34 (seeTable 1). Samples with multiple, narrow, well-defined peakscovering a very broad wavelength region are the best candidatesfor reference materials of this type. For some samples, e.g., atomiclamps, reference values of wavelengths have been certified bynational metrology institutes. For others, these values have beenestablished in the literature, e.g., a Dy-YAG crystal. Then, thereare some samples whose values have not been established, eventhough they are sold as “wavelength standards”, e.g., anthracenein PMMA. Even samples with unestablished values can be usedfor wavelength accuracy determinations, if their values arepredetermined by the user, using one of the established methods.

Linearity of the Detection System. The responsivity ofdetection systems is not linear with signal intensity at all intensitylevels. High intensity levels are most problematic because thedetector may become saturated, causing potentially large devia-tions from linear behavior. This represents one of the largestsources of uncertainty for fluorescence measurements. Linearity,as well as spectral correction (see next section), is particularlyimportant for quantification of analyte concentration from mea-sured fluorescence intensities and for applications where intensityratios 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 arange of fluorophore concentrations.19 The last of these isgenerally the easiest to implement.

Spectral Correction. Correction of detection system respon-sivity as a function of emission wavelength enables true instru-

ment-independent emission spectra to be obtained. Typicaldeviations 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 theimportance of spectral correction. An uncorrected spectrum canhave significantly different peak ratios (see Figure 1a), shape, peak

(29) Certificate of Analysis, Standard Reference Material 2065 Ultraviolet-Visible-Near-Infrared Transmission Wavelength Standard; National Institute ofStandards 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 OxideSolution; 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; HORIBAJobin 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.; Schonenberger, B.; Nording, P. J.Fluoresc. 2005, 15, 315–336.

(39) Gaigalas, A. K.; Wang, L.; He, H.-J.; DeRose, P. C. J. Res. NIST 2009, 114,215–228.

Table 1. Summary of Methods for Determining Wavelength Accuracy

sample λ region uncertainty limitations established values refs

pen lamp UV-NIR (EM) ±0.1 nm or better alignment Y 18Dy-YAG crystal 470-760 nm (EM) ±0.1 nm Y 27

255-480 nm (EX)Eu glass 570-700 nm (EM) ±0.2 nm N 28

360-540 nm (EX)anthracene in PMMA 380-450 nm (EM) ±0.2 nm limited λ range N

310-380 nm (EX)Ho2O3 + diff-use reflector 330-800 nm (EM or EX) ±0.4 nm need blank Y 29-31Xe source 400-500 nm (EX) ±0.2 nm limited λ range, calibration N 32Xe source + diffuse reflector UV-NIR ±0.2 nm monochromator must be calibrated Y 32Water raman UV-blue ±0.2 nm monochromator must be calibrated N 33, 34

Figure 1. Comparison of a spectrally corrected ( · · · · ) and anuncorrected (s) fluorescence emission spectrum for (a) standardreference material (SRM) 2941, green emission standard, taken ona fluorescence spectrometer with a monochromator/photomultipliertube based detection system (Horiba Jobin Yvon) and 427 nm lampexcitation and (b) SRM 2940, orange emission standard, taken on afluorescence spectrometer with a grating/CCD based detectionsystem (Horiba Jobin Yvon) and 404 nm laser excitation.39

2131Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

locations, and extraneous peaks (see Figure 1b) from that of thetrue spectrum. Common methods for achieving this spectralcorrection include those using a CS,32,37,38,40-42 certified emissionstandards (certified reference materials, CRMs),3-7,22,43-46 or acalibrated detector (CD) followed by a calibrated reflector(CR)32,40,41 (see Table 2). The first two methods place thecalibration tool at the sample position with light collected by theinstrument’s detection system in a one-step process. The lastmethod uses a CD to calibrate the instrument’s spectral irradiance(excitation beam power per unit area per unit bandwidth) at thesample position, followed by a CR to reflect the excitation beaminto the instrument’s detection system, in a two-step process. Allof these calibration tools are commercially available with certifiedvalues, which are SI-traceable. For instance, a CS is traceable tothe spectral irradiance scale with certified values given in SI unitsof W m-3 or common units of µW cm-2 nm-1. To judge thequality of a calibration tool, its shelf life, or recommendablerecalibration interval, the procedure used to certify or charac-terize it and its corresponding uncertainties must be stated bythe supplier.

Correction of the spectral irradiance reaching the sample as afunction of excitation wavelength enables true excitation spectrato be obtained. Many commercial instruments have their ownreference detector, which can be used for this purpose, inprinciple, if calibrated for spectral responsivity with knownuncertainties. However, a reference detector is typically designedto only account for fluctuations in the intensity of the excitationlight. A CD,32,37,38 or alternatively a quantum counter,42,47 shouldbe used for this type of spectral correction or reference detectorcalibration (see Table 3). A CD with certified values is the most

reliable choice over the broadest wavelength range. Care has tobe taken with the use of a quantum counter,42,47-49 because ofits limited wavelength range, dye concentration dependence, andpolarization dependence in viscous solvents. Similar to a CD, thespectral range and accuracy of a quantum counter, when usedfor spectral correction, should be known and not assumed.

Day-to-Day and Instrument-to-Instrument Intensity. Per-formance validation and verification standards are the most widelyneeded and requested standards for fluorescence instruments.They ensure the day-to-day consistency of measurements andshould give a constant fluorescence intensity over time under thesame experimental conditions, preferably, matching the measure-ment geometry and format (shape) of common samples, e.g, acuvette or a 96-microwell plate. Unlike many conventional stan-dards, such as calibrated light sources or calibrated detectors,they can be easily used by nonexperts. Commercially availablesolid and liquid samples, mainly in cuvette format,50,51 have beenrecommended for use as day-to-day intensity standards, and thereare a variety of advantages and disadvantages related to each. Forexample, solid samples tend to be more photostable and robust,and liquid samples are more easily adaptable to different sampleformats. In principle, such standards do not need to be certified,yet their long-term stability (and related uncertainties) needs tobe known; or for single use standards, the uncertainties introducedby the reproduction of the standard need to be known. However,laboratories working in regulated areas or accredited laboratoriestypically prefer certified standards in conjunction with evaluatedstandard operating procedures. There is a growing need for thesestandards in a microarray format.13,52-54

Day-to-day intensity standards can also be employed tocompare fluorescence intensities between different instruments,particularly when the instruments being compared share a similaroptical design. This application requires identical measurementparameters be used, e.g., the same instrument settings and sampletemperature. Users should note that signal intensity dependent

(40) Roberts, G. C. K. Correction of Excitation and Emission Spectra. InTechniques in Visible and Ultraviolet Spectrometry; Standards in FluorescenceSpectrometry; Miller, J. N., Ed.; Chapman and Hall: New York, 1981; Vol.2, Chapter 7, pp 54-61.

(41) Costa, L. F.; Mielenz, K. D.; Grum, F. In Optical Radiation Measurements;Measurement of Photoluminescence; Mielenz, K. D., Ed.; Academic Press:New York, 1982; Vol. 3, pp 139-174.

(42) Hofstraat, J. W.; Latuhihin, M. J. Appl. Spectrosc. 1994, 48, 436–447.(43) Gardecki, J. A.; Maroncelli, M. Appl. Spectrosc. 1998, 52, 1179–1189.(44) Velapoldi, R. A.; Tonnesen, H. H. J. Fluoresc. 2004, 14, 465–472.(45) Monte, C.; Resch-Genger, U.; Pfeifer, D.; Taubert, R. D.; Hollandt, J.

Metrologia 2006, 43, S89–S93.(46) Pfeifer, D.; Hoffmann, K.; Hoffmann, A.; Monte, C.; Resch-Genger, U. J.

Fluoresc. 2006, 16, 581–587.(47) Demas, J. N. In Optical Radiation Measurements, Measurement of Photo-

luminescence; Mielenz, K. D., Ed.; Academic Press: New York, 1982; Vol.3, pp 195-248.

(48) Quantum counters are highly concentrated dye solutions that transformabsorbed photons with an excitation wavelength-independent constantquantum yield into emitted photons. Such materials are prone to concentra-tion, polarization, and geometry effects resulting in enhanced calibrationuncertainties.

(49) Hart, S. J.; Jones, P. J. Appl. Spectrosc. 2001, 55, 1717–1724.(50) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Blackburn, D. H.; Kramer,

G. W. J. Lumin. 2009, 129, 349–355.(51) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Blackburn, D. H.; Kramer,

G. W. J. Lumin. 2008, 128, 257–266.

Table 2. Summary of Methods for Determining the Spectral Responsivity of the Detection System (EmissionCorrection)

sample λ region uncertainty limitations certified values refs

calibrated light source UV-NIR <±5% difficult setup Y 32, 38, 40-42calibrated detector + calibrated reflector UV-NIR ±10% difficult setup Y 32, 40, 41certified reference materials UV-NIR ±5% Y 3-7, 22

Table 3. Summary of Methods for Determining the Spectral Correction of the Excitation Channel

sample λ region uncertainty limitations certified values refs

calibrated Si photodiode UV-NIR ±2% difficult setup Y 32, 37quantum counter UV-NIR ±5% limited λ range N 42, 47Si photodiode UV-NIR e±50% N 32

2132 Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

parameters include emission spectral bandwidth, excitation spec-tral bandwidth, integration time, scan speed, and gain or voltageof detectors.

Limit of Detection. The limit of detection of an instrumentfor a particular analyte often needs to be known to establish thelowest concentration of analyte that can be detected on thatinstrument or for comparing the sensitivities of different instru-ments. Methods used to produce calibration curves for concentra-tion or to verify intensity are often used to determine sensitivityand limits of detection. Specifically, the measured fluorescencesignal of the sample is compared to the background signal of theinstrument or a blank. For example, a typical limit of detectionfor quinine sulfate dihydrate on conventional fluorescence spec-trometers is on the order of 1 ppb or 1 nM.20

Fluorescence Quantum Yield. The fluorescence quantumyield is an intrinsic property of a fluorophore that quantifies theamount of fluorescence photons emitted per amount of excitationphotons absorbed. In the new ASTM fluorescence guide, relativeand absolute optical methods for determining fluorescencequantum yields of optically dilute and optically dense samplesusing a fluorescence spectrometer are discussed.47,55,56 Relativeoptical methods, which use the fluorescence quantum yield of aknown or reference sample to determine that of an unknownunder identical measurement conditions, are much easier toimplement than absolute methods. The uncertainty of the deter-mination of the fluorescence quantum yield with a relative methoddepends on the availability of well established quantum yieldstandards and on the reliability of their fluorescence quantumyields. However, in many cases, absolute methods have to be usedbecause very few quantum yield standards have been established.

The quantum yield of a fluorophore can be highly dependenton its microenvironment, e.g., polarity, pH, temperature, oxygenconcentration, and whether or not the fluorophore is bound toanother species. This apparent interference can be used as anasset by giving insight into the characterization of differentchemical and biological microenvironments based upon measuredquantum yield values. This and other factors have contributed toa renewed interest in the measurement of fluorescence quantumyields, as attested by the recent release of new instrumentsdesigned to measure absolute quantum yields.57,58

Fluorescence Anisotropy Standards. Fluorescence anisot-ropy standards are used to calibrate or verify the performance ofinstruments that measure polarization or anisotropy of fluores-cence. Such standards should have a known anisotropy at a setor range of specified excitation and emission wavelengths. Inaddition, they should cover the anisotropy (r) range from 0.0 to0.4. Isotropic emitters (r ) 0) are also useful for measuringG-factors, i.e., the ratio of the responsivities of a detection systemto vertically versus horizontally polarized light.

Fluorescence Lifetime Standards. Samples with a singleexponential fluorescence decay and a corresponding, well-established fluorescence lifetime are used as lifetime standards.59

Their lifetimes should be independent of excitation and emissionwavelengths and have values on the same order of magnitude asthose of samples being measured. Expert laboratories recentlymeasured the lifetimes of such standards to better establish theirvalues.60 Differences between measured and established valuescan be used to correct for instrument bias.

CONCLUSIONThe new ASTM Standard Guide for FluorescencesInstrument

Calibration and Qualification gives users of fluorescence instru-ments a concise and highly informative reference, covering themajor topics and concerns involved in fluorescence detection andcalibration of fluorescence instruments. Many commonly usedcalibration methods are explained and achievable levels of ac-curacy are reported. Enough information is given about eachmethod to determine which is most likely to satisfy the needs ofa user and what materials and level of difficulty are involved. It isgeared for nonexperts, but also gives many references where userscan find more in-depth coverage.

ACKNOWLEDGMENTWe would like to thank the members of ASTM subcommittee

E13.01 Ultraviolet, Visible and Luminescence Spectroscopy fortheir contributions to the ASTM Fluorescence Guide.

Received for review November 3, 2009. Accepted January22, 2010.

AC902507P(52) Zou, S.; He, H.-J.; Zong, Y.; Shi, L.; Wang, L. In Standardization inFluorometry: State-of-the Art and Future Challenges; Springer Series onFluorescence; Resch-Genger, U., Ed.; Springer-Verlag GmbH: BerlinHeidelberg, 2008; Vol. 6.

(53) Zong, Y.; Wang, L.; Zhang, S.; Shi, L. In Microarrays Methods andApplications-Nuts & Bolts; Hardiman, G., Ed.; DNA Press, 2003; p 99.

(54) Wang, L.; Gaigalas, A. K.; Satterfield, M. B.; Salit, M.; Noble, J. InMicroarrays: Methods and Protocols, 2nd ed.; Rampal, J. B., Ed.; HumanaPress Inc., 2007.

(55) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991–1024.(56) Grabolle, M.; Spieles, M.; Gaponik, N.; Lesnyak, V.; Eychmuller; Resch-

Genger, U. Anal. Chem. 2009, 81, 6285–6294.(57) This includes an integrating sphere-based instrument by Hamamatsu and

integrating sphere-based accessories for Horiba Jobin Yvon and Edinburghfluorometers.

(58) Certain commercial equipment, instruments, or materials are identified inthis paper to foster understanding. Such identification does not implyrecommendation or endorsement by the National Institute of Standardsand Technology or the Federal Institute for Standards and Technology,nor does it imply that the materials or equipment identified are necessarilythe best available for the purpose.

(59) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer:New York, 2006; pp 883, 98, 158.

(60) Boens, N.; Qin, W. W.; Basaric, N.; Hofkens, J.; Ameloot, M.; Pouget, J.;Lefevre, J. P.; Valeur, B.; Gratton, E.; Vandeven, M.; Silva, N. D.;Engelborghs, Y.; Willaert, K.; Sillen, A.; Rumbles, G.; Phillips, D.; Visser,A.; van Hoek, A.; Lakowicz, J. R.; Malak, H.; Gryczynski, I.; Szabo, A. G.;Krajcarski, D. T.; Tamai, N.; Miura, A. Anal. Chem. 2007, 79, 2137–2149.

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