Narrow-Band Emitting Solid Fluorescence Reference Standard with

Jun 16, 2015 - (1, 2) In contrast to other optical methods like photometry, ... stability, stress tests, and on the certification procedure according ...
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Narrow-Band Emitting Solid Fluorescence Reference Standard with Certified Intensity Pattern Katrin Hoffmann, Monika Spieles, Wolfram Bremser, and Ute Resch-Genger* BAM Federal Institute for Materials Research and Testing, Division 1.10, Richard-Willstaetter-Strasse 11, D-12489 Berlin, Germany S Supporting Information *

ABSTRACT: The development of a lanthanum-phosphate glass doped with several rare-earth-ions for use as solid fluorescence standard is described. The cuvette-shaped reference material which shows a characteristic emission intensity pattern upon excitation at 365 nm consisting of a multitude of relatively narrow emission bands in the wavelength region between 450 and 700 nm is intended for the day-to-day performance validation of fluorescence measuring devices. Evaluation of the fluorescent glass includes the determination of all properties which can affect its relative emission intensity profile or contribute to the uncertainty of the certified values like absorption spectra, fluorescence anisotropy, excitation wavelength, and temperature dependence of the spectroscopic features, homogeneity of fluorophore distribution, and photo- and long-term stability. Moreover, a certification procedure was developed including the normalization of the intensity profile consisting of several narrow emission bands and the calculation of wavelength-dependent uncertainties. Criteria for the design, characterization, and working principle of the new reference material BAM-F012 are presented, and possible applications of this ready-to-use fluorescence standard are discussed.

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broad-band emitting spectral standards,12−17 as well as technical notes and recommendations for the characterization of photoluminescence measuring instruments, guides to accurate measurements of luminescence data5,7,10,11,18−23 and few recommendations on fluorescence standards.24−28 Nevertheless, there is still an urgent need for long-term stable fluorescence intensity standards for the ultraviolet, visible, and near-infrared wavelength region for monitoring instrument long-term stability and validating day-to-day instrument performance (IPV), which can be only partly covered with current certified fluorescence standards. Especially attractive is a straightforward “all-in-one solution” suitable for different fluorescence techniques providing several parameters and minimizing costs, as well as measurement time. This encouraged us to develop a new solid fluorescence standard showing a multitude of narrow-band emissions in the wavelength region between 450 and 700 nm to complement the existing glass-based and polymer-based emission standards12−17,29 for the determination of the spectral responsivity. For this purpose, we evaluated a very robust fluorescent glass doped with several highly emissive rare earth metal ions. The present report on the spectroscopic features of this solid reference material also details information on the homogeneity of fluorophore concentration, long-term stability, stress tests,

hotoluminescence methods are among the most commonly used analytical techniques in the life and the material sciences due to their straightforwardness, sensitivity, and nondestructive character. Fluorescence-based techniques can provide several analyte-specific properties. i.e. emission and excitation spectra, luminescence quantum yields, luminescence lifetimes, and emission anisotropies. Their potential for combining spectrally, temporally, and spatially resolved measurements open up advanced possibilities for many multiplexing applications and enable remote sensing.1,2 In contrast to other optical methods like photometry, all photoluminescence signals, however, include undesired wavelength-, polarization-, and time-dependent impacts due to instrument-related effects and are hence affected by aging of optical and electro-optical device components. Furthermore, substantial challenges to measure absolute luminescence intensities3−6 complicate the comparison of data recorded with different instruments. These problems can be smartly resolved with fluorescence standards that enable an instrument characterization under application-relevant conditions and signal referencing. A first step to the eventually desired standardization of fluorescence measurements presented the spectral emission standards F001−F005, developed according to our concept of method-adaptable reference materials, enabling their use for different fluorescence techniques, sample formats, and measurement geometries.7−9 The performance of the standard set was assessed in an interlaboratory comparison of several National Metrology Institutes.10,11 Nowadays, there exist some more © 2015 American Chemical Society

Received: March 23, 2015 Accepted: June 16, 2015 Published: June 16, 2015 7204

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Analytical Chemistry and on the certification procedure according to ISO Guides 34 and 35.37 Moreover, examples for its applicability and limitations of its use are given.

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r=

I − I⊥ I + 2I⊥

(1)

Photostability. Photostability studies were performed with randomly chosen slide-shaped glass samples obtained from the same glass batch as BAM-F012. These studies were carried out with a custom-built setup designed for photostability studies using an excitation wavelength λexc = 365 nm as employed for certification measurements and recommended for use of BAMF012. The radiant power density of 0.688 mW/cm2 at the sample position was at least 1 order of magnitude higher than that used for typical fluorescence measurements.

EXPERIMENTAL SECTION

Candidate Reference Material. The material used for the preparation of the certified reference materials (CRM) BAMF012 is a fluorescent glass doped with several rare earth (RE) metal ions. The custom-designed glass containing 3.71 wt % Eu203, 2.62 wt % Tb203, and 0.12 wt % Ce203 was obtained from Schott AG, Germany, and has been previousy detailed.8,30−33 All candidate reference glass blocks studied originate from three glass melts (melt volumes 3 × 2 L) of identical elemental composition. These batches were cut into 60 individual pieces. The final reference material consists of a rectangular block of solid multiemitter (ME) glass with typical cuvette dimensions (12.5 mm × 12.5 mm × 40 mm) and four polished long faces. Spectrofluorometry. All fluorescence measurements were performed with a spectrofluorometer SLM 8100 (Spectronic Instruments, Peltier cooled PMT (R928, Hamamatsu)) in a 0°/ 90° measurement geometry with an integration time of 1 s in the photon counting mode. The Glan-Thomson excitation polarizer was set to 0° and the emission polarizer to 54.7° (magic angle conditions), respectively, if not stated otherwise, e.g. for the anisotropy measurements. The calibration of the spectrofluorometer has been described earlier.4 During spectra recording, the spectral radiant flux of the excitation channel was always monitored with a reference detector that accounts for its short-term fluctuations. The certified relative emission intensity pattern of the glass-based multiemitter fluorescence standard BAM-F012 was obtained from spectrally corrected emission spectra excited at 365 nm (excitation slit of 4/8/4) and detected with an emission slit set to 2/4/2 using under magic angle conditions. The notation of the monochromator slit widths corresponds to entrance slit/ center slit/exit slit of the double monochromators, in the emission and excitation channel. For the correction of the measured relative fluorescence intensities for instrument- and wavelength-dependent spectral characteristics, the previously determined relative values of the spectral responsivity s(λ) of the spectrofluorometer were used. This ensures the metrological traceability of the emission intensity pattern to the spectral radiance scale.3,4,10 Temperature-Dependent Measurements. To assess temperature effects, the emission spectra of BAM-F012 were measured between −7.3 and 50 °C. Temperature control was achieved using a thermostated cuvette holder operated with a water/ethylene glycol mixture (4:1). In the temperature range of interest, heating, as well as cooling, cycles were performed. The systems were allowed to thermally equilibrate for 15 min. Measurements were performed in a nitrogen stream to prevent condensation at lower temperature. Anisotropy Measurements. Glan-Thomson polarizers were used to measure the fluorescence intensities with the excitation polarizer set to 0° and the emission polarizer to 0° (parallel ∥) and 90° (perpendicular ⊥), respectively. The resulting polarization-dependent raw spectra were spectrally corrected, and the wavelength-dependent intensities were then used to calculate the emission anisotropy r according to

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RESULTS AND DISCUSSION Optical Properties. The emission spectra of the RE-doped fluorescent glass material show a multitude of relatively sharp emission bands in the wavelength region between 450 and 720 nm upon excitation at 365 nm as highlighted in Figure 1. Eight

Figure 1. Assignment of the evaluated absorption of the multiemitter glass (A), the measured fluorescence emission spectrum (excitation at 365 nm, intensity at 612 nm ca. 200 000 counts, measured with spectrofluorometer SLM 81000 equipped with a red sensitive PMT R928 (Hamamatsu); integration time 1 s, magic angle conditions) together with the inverse spectral responsivity s(λ) (solid circles) of the instrument used (B) and the effect of spectral correction (C).

characteristic emission bands or shoulders (sh) at 488 nm (Tb3+: 5D4 → 7F6), 542 and 548 nm (sh) (Tb3+: 5D4 → 7F5), 591 nm (Tb3+: 5D4 → 7F4/ Eu3+: 5D0 → 7F1), 612 nm (Eu3+: 5 D0 → 7F2), 620 nm (sh) (Tb3+: 5D4 → 7F3), 653 nm (Eu3+: 5 0 D → 7F3), and 701 nm Eu3+: (5D0 → 7F4) were evaluated. Normalization. To compare the results of emission measurements of the fluorescence standards, a simple, clear, and easily reproducible normalization procedure was developed, chosen from several normalization alternatives, including the individual intensities of the eight peaks in the emission profile, the area of all peaks (integral emission curve) and the sum of the intensities of the eight peaks. The normalization procedure based on the sum of the individual peak intensities at previously defined wavelengths turned out to be best suited for our purpose (see Figure S-1; Supporting Information SI) and was subsequently used for data evaluation. Homogeneity of the Samples. All candidate reference materials originate from three glass melts of identical composition. The spectroscopic properties of randomly selected glass cuvettes from these batches reveal an excellent spatial homogeneity indicating a homogeneous dopant distribution and are nearly identical for different samples (see 7205

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were observed, underlining the material’s high photostability (see Figure S-5; SI). Therefore, the results of the photostability study were not taken into account in the calculation of the uncertainty budget. Polarization Effects. Fluorescence measurements can be affected by instrument- and sample-specific polarization effects. Device-specific effects include the degree of polarization of the exciting spectral radiant flux reaching the sample and the polarization-dependent spectral sensitivity of the detection system, i.e. the ratio of its spectral sensitivity to horizontally and vertically polarized light.3 These effects are illustrated in Figure 3 (left), showing variations in measured emission spectra of the

Figure S-2; SI). A few of the studied glass blocks show clearly visible striae due to spatial variations of the refractive index. The band-like striae do not exhibit sharp edges, and their shape is similar to a frozen convection pattern. The inhomogeneities arising from differences in refractive index between the striae and the remaining material are in the range of about ∼10−6 and barely influence the standard-relevant optical properties.36 Statistical evaluation of the normalized peak intensities using one-factorial analysis of variance (ANOVA) only revealed a significant between-unit difference for the double peak 542/548 nm but are still homogeneous at a 1% significance level. The material can be considered sufficiently homogeneous according to the requirements of ISO Guide 35. Furthermore, all homogeneity contributions to the measurement uncertainty are duly calculated and taken into account in the total uncertainty budget of the certified values. The relative standard uncertainties due to sample inhomogeneity ubb,r account to values within 0.2% and 1% depending on the different emission bands. Excitation Wavelength Dependence. An ideal fluorescence standard should have a constant fluorescence quantum yield and an emission profile independent of excitation wavelength. Hence, the emission spectra of several glass samples were examined using predetermined measurement parameters but varying excitation wavelengths in the range between 360 nm and 370 nm with increments Δλ = 1 nm. This wavelength was chosen as the most common excitation sources (Xe/Hg lamp) favor an application at 365 nm. As to be expected from the absorption spectra shown in Figure 1A, all evaluated emission bands reveal a more or less strong dependence on excitation wavelength. The most pronounced effects are observed for the double peaks at about 545 nm and peaks at 612 and 620 nm (see Figure 2), respectively.

Figure 3. Measured, polarization-dependent fluorescence spectra (red: polarizer setting 0°, 0° and black: 0°, 90°) (left). Spectrally corrected curves (right) reveal almost no emission anisotropy. Excitation was at 365 nm.

multiemitter glass recorded with different polarizer settings. Almost all monochromator-based fluorescence measurement systems excite the fluorescence with partially polarized light unless a depolarizer is used which generates nonpolarized light from the excitation source. Hence, standards with a highly isotropic emission are preferred for the determination of the spectral characteristics of fluorescence measuring systems as they can be used without polarizers. A measure of the degree of emission polarization of fluorescent materials is the anisotropy r of their emission. Fluorescence standard materials should be preferably isotropic emitters with r ≤ 0.05. The emission anisotropy of several glass standards was assessed using previously specified measurement parameters (see Experimental Section), yet recorded with different polarizer settings. The excitation polarizer was set to 0° and the emission polarizer either to 0° (vertically polarized) or 90° (horizontally), respectively. To calculate the emission anisotropy r, the sum of the data points of the corresponding spectrally corrected emission profiles represented in Figure 3 were used for the intensities in eq 1. As all emission bands show r values between 0 and 0.05 (see Table S-2 (SI)) the relative uncertainty contribution uaniso resulting for polarization effects accounts to values between 0.1% and 6% depending on the respective peak. Temperature Effects. Fluorescence is a temperaturesensitive quantity.1 To assess the influence of temperature on the spectroscopic properties of the multiemitter glass, the absorption and emission spectra of the glass blocks were studied in an application-relevant temperature range between −7.3 and 50 °C with increments of 10 K. The data obtained from heating and cooling cycles showed almost no temperature effect on peak positions, shapes, and intensities. The integral emission derived from the spectra is depicted in Figure 4, together with the corresponding linear fit.

Figure 2. Deviations of normalized emission intensities caused by variations in excitation wavelength.

Variation of the recommended excitation wavelength λexc = 365 ± 1 nm leads to significant deviations from the certified values as shown in Figure 2. Uncertainty contributions uexc due to variation in excitation wavelengths are summarized in Table S-1 (SI). Photostability. To simulate their frequent use, the photostability of the glass was tested using a custom-built photostability setup. The spectral radiant flux was chosen to be at least 1 order of magnitude higher than in typical fluorescence measurements. Even after 17 h of illumination no changes in photocurrent, which is proportional to the optical transmission, 7206

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Figure 4. Mean of the integral fluorescence emission intensities (integration from 450 to 720 nm) at different temperature for the heating and cooling cycle, the corresponding linear fit, and the boundaries of the 95% confidence interval, respectively.

Figure 5. Relative corrected and normalized fluorescence intensities of the eight band maxima of samples kept at different storage conditions: room temperature 25 °C (RT), refrigerator 8 °C (KS), freezer −18 °C (TS), and heating chamber 70 °C (HS).

The data points vary randomly and do not show any trend. Moreover, a slope close to zero (8.53 × 10−5 ± 8.96 × 10−4) indicates almost no effect of short-term temperature variation on emission profiles in the temperature range studied. Long-Term Stability. An accelerated aging test to simulate the normal aging process was performed with 12 randomly selected fluorescence standard samples. Different stress levels with storage conditions (SC) at various temperatures, measured in time intervals of 0, 2, 4, 8, 16, and 21 weeks, have been used to obtain information on long-term effects. Typically 3 samples were used for each storage condition including −18 °C (freezer (TK)), +8 °C (refrigerator (KS)), room temperature (25 °C) in an air-conditioned room (RT)), and +70 °C (drying cabinet (temperature typically (70 ± 3 °C)). The periodically measured absorption, as well as the emission spectra, showed no differences in band position and only very minor changes in intensity of few bands (see Figure S-6; SI). In order to assess thermal effects and to estimate the expected shelf life of the fluorescence standard the relative standard deviations Sr of the band intensities measured at different predefined maturity dates of the campaign (see Figure S-7; SI) were examined. To circumvent an influence of instrument aging, the measurements were performed following an isochronous scheme.37 The largest deviation was found for the band centered at 653 nm, most likely because of its low signal-to-noise-ratio. The relative intensities It=0/It=x of the corrected and normalized fluorescence intensities of the emission maxima under study showed a maximum variation of ±1% depending on different storage times, leaving no discernible trend. Also the comparison of the four storage conditions (see Figure 5) revealed no significant drift. Striking was again the weak emission band at 653 nm. Therefore, this band was not used in the following data assessment. To estimate the long-term stability of the multiemitter standard, the variations in SC-dependent intensities of all eight emission bands were evaluated. The corresponding slopes of the linear regression lines over storage time were plotted against the inverse storage temperature (1/T in K−1). As follows from the data displayed in Figure 6, both the RT data and the slopes for the stress levels KS, TK, and HS are around zero for all bands with maximum values found for the peak at 653 nm. From the results of this aging study and from a worst case estimation, we concluded that the material is insensitive to storage condition changes. Nevertheless an

Figure 6. Stress test for stability assessment of BAM-F012. Dependence of the slopes of the regression lines on 1/T.

exposure to the highest and lowest temperatures over a period of time of 1 week or more should be avoided. From long-term studies over about 72 months, a shelf life of the fluorescence standard of 7 years has been estimated (see Figure S-10, Figure S-11, and Table S-3; SI). An additional contribution ults to the uncertainty budget had to be made only for the peak at 591 nm, which degrades slightly faster than other peaks. Certified Values and Traceability. The certified values of the emission pattern of BAM-F012, excited at 365 nm, and their expanded combined uncertainties derived from the normalized relative emission intensities of the eight narrow bands determined relative to spectral radiance scale, are shown in Figure 7. The uncertainties of the corrected emission spectra combine the previously determined relative uncertainties of the calibration of the BAM fluorometer4 used for certification ucal and the relative uncertainties of the measurements of the emission spectra uchar, as well as material-related uncertainties. The latter are derived from sample homogeneity (ubb) and stability studies (ults), as well as from the excitation wavelength dependence uexc and the fluorescence anisotropy uaniso, respectively. The results of the certification study were evaluated in full accordance with ISO Guide 35.19,37 The combined uncertainty ucombined was calculated according to eq 2 where ui are the different contributions described before 7207

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Monitoring of Spectrofluorometer Performance. Provided a strict control of excitation wavelength accuracy and the use of always identical instrument settings, BAM-F012 is suitable to monitor temporal changes of the wavelength-dependent relative spectral responsivity s(λ) of fluorescence instruments, indicative of the aging of optical and optoelectronic instrument components. This can help to render instrument calibration more efficient. Knowledge of s(λ) is the prerequisite for the accurate correction of emission spectra for instrument-specific effects.3,4,10,11,24,34,35 Comparison of the measured emission intensity pattern with the certified values of the emission bands allows evaluation of the reliability of the calibration of the emission channel of spectrofluorometers. This includes the wavelength accuracy of the excitation and the emission channel, as well as changes in s(λ). This is, however, restricted to instruments with continuous excitation sources. Thus, the fluorescent standard is well suited to rule out instrumentation effects and to enable instrument-independent, thus comparable fluorescence data. Results of exemplary assessments of the new calibration tool are depicted it Figure 8, underlining its potentials to communicate erroneous measurements or incorrect data handling by deviations from the certified emission pattern.

Figure 7. Emission intensity pattern of BAM-F012 from the spectrally corrected and normalized emission spectrum; excitation was at 365 nm. Uncertainties using a coverage factor of k = 2 correspond to a confidence interval of approximately 95%. The certified values are based on the results of 512 independent measurements on 24 cuvetteshaped glass samples.

ucombined =

2 2 2 2 2 ucal + uchar + ubb + ults2 + uexc + uaniso

(2)

As the certified intensity pattern of BAM-F012 is based on spectrally corrected emission spectra, measured with the calibrated BAM reference spectrofluorometer SLM 8100, the metrological traceability of the emission intensity pattern to the spectral radiance scale is ensured.4 Application and Recommended Use. Day-to-Day Intensity Standard. BAM-F012 is intended to serve as a standard for Instrument Performance Validation (IPV) of fluorescence measurement devices using identical measurement conditions to periodically assess their day-to-day-performance. Signal-intensity affecting parameters to be kept constant include the excitation wavelength, spectral excitation and emission bandwidths, polarizer settings, gain or voltage of the detectors, scan speed, and integration time. The regular evaluation of variations of the emission intensity pattern can either be based on the raw spectra or the normalized emission intensities derived from uncorrected emission spectra of BAM-F012. Use of BAM-F012 as an instrument-to-instrument intensity standard is recommended for instruments with a continuous excitation source since the different rare earth ions in BAMF012 have comparably long dopant-specific emission lifetimes in the microsecond to millisecond region. Therefore, special care has to be taken for use of this multiemitter-doped glass for the characterization of instruments equipped with pulsed light sources. In this case, the RE glass can only be applied as day-today intensity standard for a fixed set of measurement parameters, e.g. pulse duration, delay, and time gate, as these parameters considerably influence the emission properties of the glass in an emission band-specific way. Validation of the Wavelength Accuracy. Based on its relatively narrow band characteristics in emission, the reference material may also be employed as a wavelength standard for the validation of the wavelength accuracy of absorption spectrometers, as well as of the emission channel of fluorescence instruments with low requirements on spectral resolution. Due to the significant excitation wavelength dependence (see above), also the long-term stability of the wavelength accuracy of excitation channel of fluorescence instruments can be assessed by regular evaluation of the emission spectrum of the RE glass and the certified emission intensity pattern.

Figure 8. Deviation from certified emission pattern due to a lack of spectral correction (open hexagons), corrected data based on photonic units instead of energy units (open triangles), and data recorded beyond the linear detector range (open squares). Properly corrected data measured with two different spectrofluorometers A (solid triangles) and B (solid squares) yield values close to the certified emission intensity pattern.

Data recorded beyond the linear range of the instrument’s detection system, e.g., result in spectral distortions due to detector saturation (open squares). Significant deviations arise also from the lack of spectral correction (open hexagons) or from inaccurately corrected raw data. This can be utilized to assess the reliability of correction curves or correction factors, which are often implemented in spectrofluorometer software. In most photon counting spectrofluorometers, the data and correction curves are provided in units of photons. This references the emission correction curve to the spectral photon radiance scale. The use of such data (open triangles in Figure 8), as well as generally inadequately corrected data, results also in deviations from the certified values. Vice versa, a good match with the certified intensity profile provides a straightforward evidence for properly corrected data measured within the linear detector range (solid squares) and referring to an instrument 7208

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regarding fluorescent glasses and M. Spieles and D. Sewohl for their support with spectroscopic measurements.

calibration referenced to the spectral radiance scale (solid triangles).

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CONCLUSION With the cuvette-shaped glass-based multiemitter glass BAMF012, with its fluorescence intensity profile certified for excitation at 365 nm, we developed a robust and traceably characterized fluorescence reference material for the evaluation of fluorescence instruments. The certified and long-term stable fluorescence standard, which has been optimized for spectrofluorometers with a 0°/90°, measurement geometry, equipped with continuous (nonpulsed) excitation sources, enables efficient Instrument Performance Validation (IPV) under routine measurement conditions. Standards for IPV are currently the most widely needed and requested standards for all types of fluorescence measuring systems. Moreover, it can detect inaccuracies in the wavelength scale. The regular application of BAM-F012 communicates changes in the wavelength-dependent spectral responsivity of the instrument via changes in the intensity pattern of its emission spectrum and provides the basis for an improved reliability and comparability of emission data over time and across instruments on a very broad level. In addition, the comparison of measured emission spectra and intensity profiles with the certified data enables the assessment of the quality of spectral corrections curves and provides hints for aging of spectrometer components and, hence, the need for instrument recalibration. The RE-glass can be used, yet with special care, for the comparison of different instruments with a continuous excitation source, which share a similar optical design. The fluorescent glass is currently evaluated with different shapes as intensity reference for use in other fluorescence applications like microplate readers or fluorescent sensors. In summary, the multiemitter fluorescence standard BAMF012 can perfectly complement existing broad-band fluorescence standards and is an ideal tool for all users of fluorescence techniques working in regulated areas like medical diagnostics or pharmaceutics, which need easy-to-handle certified standards for use under routine measurement conditions in conjunction with validated standard operating procedures.

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(1) Lakowicz, J. R. Principles of fluorescence spectroscopy, 3rd ed.; Springer Science+Business Media, LLC: New York, 2010. (2) Topics in Fluorescence Spectroscopy Vol. 1−8; Lakowicz, J. R., Ed.; Plenum Press: New York, 1992−2004. (3) 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. (4) Hollandt, J.; Taubert, R. D.; Seidel, J.; Resch-Genger, U.; GuggHelminger, A.; Pfeifer, D.; Monte, C.; Pilz, W. J. Fluoresc. 2005, 15, 301−313. (5) Resch-Genger, U.; Pfeifer, D.; Hoffmann, K.; Flachenecker, G.; Hoffmann, A.; Monte, C. In Standardization and Quality Assurance in Fluorescence Measurements I: Techniques; Resch-Genger, U., Ed.; Springer Verlag: Berlin-Heidelberg, 2008; pp 65−100. (6) Resch-Genger, U.; Hoffmann, K.; Nietfeld, W.; Engel, A.; Neukammer, J.; Nitschke, R.; Ebert, B.; Macdonald, R. J. Fluoresc. 2005, 15, 337−362. (7) Resch-Genger, U. Standardization and Quality Assurance in Fluorescence Measurements II Bioanalytical and Biomedical Applications, 1st ed.; Springer-Verlag: Berlin, Heidelberg, 2008; p 1−560. (8) Hoffmann, K.; Monte, C.; Pfeifer, D.; Resch-Genger, U. GIT Lab. J. 2005, 6, 29−30. (9) Hoffmann, K.; Resch-Genger, U.; Nitschke, R. In Standardization and quality assurance in fluorescence measurements II - Bioanalytical and biomedical applications 6 Part A; Resch-Genger, U., Ed.; Springer: 2008; pp 89−116. (10) Resch-Genger, U.; Bremser, W.; Pfeifer, D.; Spieles, M.; Hoffmann, A.; DeRose, P. C.; Zwinkels, J. C.; Gauthier, F.; Ebert, B.; Taubert, R. D.; Monte, C.; Voigt, J.; Hollandt, J.; Macdonald, R. Anal. Chem. 2012, 84, 3889−3898. (11) Resch-Genger, U.; Bremser, W.; Pfeifer, D.; Spieles, M.; Hoffmann, A.; DeRose, P. C.; Zwinkels, J. C.; Gauthier, F.; Ebert, B.; Taubert, R. D.; Voigt, J.; Hollandt, J.; Macdonald, R. Anal. Chem. 2012, 84, 3899−3907. (12) DeRose, P. C.; Smith, M. V.; Anderson, J. R.; Kramer, G. W. J. Lumin. 2013, 141, 9−14. (13) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Anderson, J. R.; Kramer, G. W. J. Lumin. 2011, 131, 1294−1299. (14) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Anderson, J. R.; Kramer, G. W. J. Lumin. 2011, 131, 2509−2514. (15) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Blackburn, D. H.; Kramer, G. W. J. Lumin. 2008, 128, 257−266. (16) DeRose, P. C.; Smith, M. V.; Mielenz, K. D.; Blackburn, D. H.; Kramer, G. W. J. Lumin. 2009, 129, 349−355. (17) Starna Scientific Limited; UK. http://www.starna.com/ ukhome/d_ref/f_ref/xflou.html. (18) 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. NCCLS, I/LA24-A 2004, 24. (19) Gaigalas, A. K.; Li, L.; Henderson, O.; Vogt, R.; Barr, J.; Marti, G.; Weaver, J.; Schwartz, A. J. Res. Natl. Inst. Stand. Technol. 2001, 106, 381−389. (20) Roberts, G. C. K. In Techniques in Visible and Ultraviolet Spectrometry; Miller, J. N., Ed.; Chapman & Hall: New York, 1981; pp 49−67. (21) Velapoldi, R. A.; Epstein, M. S. In Luminescence Applications in Biological, Chemical, Environmental, and Hydrological Sciences; Goldberg, M. C., Ed.; American Chemical Society: WA, 1989; pp 98−126. (22) Gaigalas, A. K.; Wang, L. L.; He, H. J.; DeRose, P. J. Res. Natl. Inst. Stand. Technol. 2009, 114, 215−228. (23) Resch-Genger, U.; DeRose, P. C. Pure Appl. Chem. 2012, 84, 1815−1835. (24) DeRose, P. C.; Resch-Genger, U. Anal. Chem. 2010, 82, 2129− 2133.

ASSOCIATED CONTENT

S Supporting Information *

Eleven supplementary figures (Figures S1−S11) and three tables (Table S1−S3) contain additional experimental data and Supporting Information, which detail the evaluation of studies on normalization procedure, excitation wavelength dependence, fluorescence anisotropy, and photo and long-term stability, as well as temperature effects. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02209.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Fax: 49 30 8104 71159. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Federal Ministry of Education and Research (13N8850). We express our gratitude to A. Engel (Schott AG) for helpful discussions 7209

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