Certified Fluorescence Standards

By: U. Resch-Genger, D. Pfeiffer, A. Hoffmann, R. Meier, A. Rück, P. Nording, B. Schönenberger, BioFiles 2007, 2.5, 19.

U. Resch-Genger, D. Pfeiffer, A. Hoffmann, Federal Institute for Materials Research and Testing (BAM), Germany
R. Meier, A. Rück, P. Nording, B. Schönenberger, Buchs, Switzerland

There is an increasing need for reliable and comparable data, quantification, and standardization for luminescence techniques that are widely used in the life sciences, material sciences and environmental analysis.1-4 Instrumentation can be a major source of variability, making comparison of fluorescence measurements between instruments and over time difficult. Standardization can be achieved with reliable standards in combination with specific protocols for instrument characterization and instrument performance validation (IPV).5

In collaboration with the Federal Institute for Materials Research and Testing (BAM, Berlin, Germany), we have developed a Spectral Fluorescence Standards Kit.6

The Spectral Fluorescence Standards Kit provides a simple and flexible calibration tool for the determination and control of the relative spectral responsiveness of fluorescence instruments. The kit is optimized for use with spectrofluorometers, provides traceability of fluorescence measurements to the spectral radiance scale, and can be used with various measurement geometries. It can be adapted for calibration of other fluorescence measuring instruments with proper consideration of the underlying measurement principles

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Instrument Effects on Fluorescence Signals

Fluorescent signals are influenced by both sample- and instrument specific effects. In practice, it is difficult to reproduce fluorescent data on different instruments or at different times with the same instrument, regardless of experimental parameters. This variance in results is due to instrument-specific contributions to the fluorescent signal. These wavelength-, polarization- and time-dependent differences reflect:

  • The spectral radiance of the excitation light source
  • The transmittance of optical components, including lenses, mirrors, filters, monochromator gratings, and polarizers in the excitation and emission channels
  • The spectral responsivity of the detection system

The influence of the spectral responsivity s(λem) on fluorescence data and the resultant need for its consideration, i.e., spectral emission correction, are illustrated for a typical organic fluorophore analyzed using two spectrofluorometers (see Figure 1).

Figure 1. Normalized uncorrected (open symbols, blue and green) and corrected (full symbols) emission spectra of a typical organic fluorophore measured with two different fluorometers.

The ability to compare fluorescence data and the use of fluorescence spectra for the identification of analytes rely on the knowledge and consideration of s(λem) and its time-dependent changes due to degradation of the instrument components.7 The latter makes regular control of s(λem) mandatory but simultaneously comprises an elegant tool for IPV and control of instrument longterm stability (see Figure 2).

Figure 2. Comparison of the typical spectral radiance of a tungsten ribbon lamp (top, red), an integrating sphere type radiator (middle, green), and typical fluorophores (bottom, blue).

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Determination of Spectral Responsivity

The spectral responsivity value of an instrument can be obtained by evaluating a reference material that emits a known and broad spectrum in the ultraviolet/visible/near infrared region.7,8 In general, three primary types of light sources can be used:

  • Lamps (e.g. tungsten ribbon lamps)
  • Sphere-type spectral radiators
  • Fluorophore standards

Tungsten ribbon lamps and integrating sphere-type spectral radiance transfer-standards are traceable, but are tedious to align, can impose restrictions on measurement geometry, require regular calibration, which can be expensive, and often do not physically fit into compact fluorescence instruments.7,8 Moreover, their spectral radiances/emission intensities exceed those of typical fluorescent samples by approximately two and four orders of magnitude, respectively, for integrating sphere radiators and tungsten ribbon lamps (see Figure 2). Accordingly, to avoid errors due to the nonlinearity of the detection system, their use requires attenuation procedures that do not introduce additional spectral effects.6

In contrast, fluorophore-based spectral fluorescence standards7,9-13 are easy to use and offer flexibility with respect to measurement geometry and instrument type. These standards can be measured under routine conditions, reducing many sources of systematic error. Fluorophore standards must be selected to encompass the ultraviolet/visible/near infrared spectral region. Each standard must also give a broad and unstructured emission spectrum to minimize the influence of spectral bandwidth on spectrum shape, and have a very small fluorescence anisotropy to minimize polarization effects and ameliorate the need for polarizers.6,7,14

Additional requirements for spectral fluorescence standards are:
(1) Traceable and accurate measurement of their corrected emission spectra with a reported uncertainty.
(2) Characterization of their calibration-relevant properties according to EN ISO/IEC 17025 and ISO Guides 34 and 35.

When properly selected, these standards can be measured under routine conditions, reducing sources of systematic error and providing the basis for comparable emission data.

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Principle and Properties

The Spectral Fluorescence Standards Kit consists of:

  • Five spectral fluorescence standards covering the spectral region of 300 to 760 nm
  • BAM-certified normalized corrected emission spectra
  • Ethanol for use as a solvent
  • LINKCORR software. The LINKCORR software was developed and evaluated by BAM for the calculation of s(λem) based on the measured and certified emission spectra of the standards provided in the kit.
  • Technical insert with calibration procedure

The emission spectra, LINKCORR software, and technical insert are provided on a single compact data disk.

The five standards have been selected to match a set of spectral properties requirements.13,14 The standards are also available separately. Working solutions of each of the fluorescent standards are measured with the instrument to be calibrated, and data is evaluated with the provided LINKCORR software (see Figure 3).

Figure 3. Working principle of the Spectral Fluorescence Standards Kit. See text for detailed explanation.

The LINKCORR software calculates the quotients QF00x(λem) of the certified emission spectra Ic(λem) (see Figure 3A, solid lines) and the uncorrected emission spectra Iu(λem) (Figure 3B, dashed lines) for each dye. As determined by the LINKCORR software, a weighted combination of QF00x(λem) (Figure 3B, solid color lines) yields an overall emission correction curve (Figure 3B, solid black line). This curve equals the inverse relative spectral responsivity of the instrument (1/s(λem)). The relative spectral responsivity of the instrument (S(λ)) is indicated in Figure 3B (broken black line). Corrected instrument-independent and comparable data are then obtained upon multiplication of measured spectra with this correction curve (1/s(λem)).

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Traceability, Certification and Proficiency Testing

The Spectral Fluorescence Standards Kit has been certified by BAM according to ISO Guidelines 34 and 35, and the standards are traceable to the spectral radiance scale.6-8,14 The wavelengthdependent relative uncertainties of the corrected emission spectra are a combination of the relative uncertainties of the calibration of the fluorometer used for certification, the measurements of the emission spectra, and material-related uncertainties derived from homogeneity and stability studies.14

The corrected emission spectra of the kit standards and the determination of an emission correction curve using the Spectral Fluorescence Standards Kit have been evaluated by sequential analysis performed by:

  • The National Metrological Institutes (NIST, U.S.A.)
  • The National Research Council of Canada (NRC)
  • The Physikalische Technische Bundesanstalt (PTB, Germany)
  • Federal Institute for Materials Research and Testing (BAM, Berlin, Germany)

The laboratories used four characterized instruments and two different measurement geometries (0°/90° and 45°/0°). In addition, the Spectral Fluorescence Standards Kit has been successfully tested with several types of common spectrofluorometers.7,14 The results depicted in Figure 4 compare the uncorrected and standard- corrected emission spectra of three test dyes X, QS, and Y. The corrected fluorescence spectra demonstrate a variance of less than 10%. This precision is similar to what is achievable by experienced technicians using physical transfer standards, tedious and time-consuming procedures, and severe geometry restrictions.7,14

Figure 4. Proficiency testing of the Spectral Fluorescence Standards Kit. Kit-based spectral correction of A) the uncorrected emission spectra of the test dyes X, QS, and Y measured with four fluorometers and B) comparison of the resulting corrected emission spectra. A variance of <10% is achieved after correction.

By combining multiple standards, convenient software analysis, and independent validation, the Spectral Fluorescence Standards Kit now allows researchers using fluorescent detection to establish comparable and traceable fluorescent measurements and to identify valid instrument performance.

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  12. Velapoldi, R.A. and Mielenz, K.D., A fluorescence standard reference material: Quinine sulfate dihydrate, NBS Spec. Publ. 260-64, PB 80132046, Springfield, VA (1980).
  13. Gardecki , J.A. and Maroncelli, M., Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy, Appl. Spectr., 52, 1179-1189 (1998).
  14. Resch-Genger, U. et al., The calibration Kit Spectral Fluorescence Standards-A simple and certified tool for the standardization of the spectral characteristics of fluorescence instruments, J. Fluoresc., 16, 581-587, (2006).
  15. Hoffmann, K., et al., Standards in Fluorescence Spectroscopy: Simple Tools for the Characterization of Fluorescence Instruments, G.I.T. Laboratory Journal, 5, 2-4 (2005).

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