Article pubs.acs.org/ac
Integrating Sphere Setup for the Traceable Measurement of Absolute Photoluminescence Quantum Yields in the Near Infrared Christian Würth, Jutta Pauli, Cornelia Lochmann, Monika Spieles, and Ute Resch-Genger* BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany S Supporting Information *
ABSTRACT: There is an increasing interest in chromophores absorbing and emitting in the near-infrared (NIR) spectral region, e.g., for applications as fluorescent reporters for optical imaging techniques and hence, in reliable methods for the characterization of their signal-relevant properties like the fluorescence quantum yield (Φf) and brightness. The lack of well established Φf standards for the NIR region in conjunction with the need for accurate Φf measurements in transparent and scattering media encouraged us to built up an integrating sphere setup for spectrally resolved measurements of absolute fluorescence traceable to radiometric scales. Here, we present the design of this setup and its characterization and validation including an uncertainty budget for the determination of absolute Φf in the visible and NIR. To provide the basis for better measurements of Φf in the spectral window from ca. 600 to 1000 nm used, e.g., for optical imaging, the absolute Φf of a set of NIR chromophores covering this spectral region are measured and compared to relative values obtained using rhodamine 101 as Φf standard. Additionally, the absolute Φf values of some red dyes that are among the most commonly used labels in the life sciences are presented as well as the absolute quantum yield of an optical probe for tumor imaging.
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methods are the accuracy of the radiometric spectrofluorometer characterization and the reliability of the standard's Φf value.4,10,23−25 This value must be precisely known for the measurement conditions employed (e.g., solvent/matrix, excitation wavelength λex, temperature, etc.).26 However, at present, Φf values of only very few fluorophores emitting in the visible (vis) region like rhodamine 101, rhodamine 6G, fluorescein, and quinine sulfate dihydrate are well established with relative uncertainties of ca. 5%, typically for especially purified dyes not commercialized.11,22,27 Although functional chromophores absorbing and emitting above 600 nm are increasingly being used in biology, molecular imaging, and clinical diagnostics,28−33 no dependable quantum yield standards for the near-infrared (NIR) spectral region are currently available.8,11 For example, in the case of the frequently employed NIR quantum yield standard indocyanine green (ICG), one of the most fundamental publication states that the Φf values provided present only trends due to questionable dye purity.34 Moreover, considerable uncertainties in the size of the Φf value of IR26 led to an overestimation of the photoluminescence quantum yields of many PbSe and PbS quantum dots determined relative to this dye as recently disclosed.8 Alternatively, the number of emitted photons Nem(λex) per number of absorbed photons Nabs(λex), see eq 1, can be measured absolutely with an integrating sphere setup, thereby
entral advances in biochemical assays, molecular sensor technologies, and molecular optical imaging are related to fluorescent reporters that provide a high detection sensitivity for molecular processes in conjunction with a high selectivity. The most fundamental spectroscopic properties of such functional chromophores are the fluorescence quantum yield Φf, the molar absorption coefficient at the excitation wavelength ε(λex), and for some applications, also the fluorescence lifetime.1−4 Of special importance is the fluorometric key parameter Φf, i.e., the number of emitted photons Nem per number of absorbed photons Nabs that presents a direct measure for the efficiency of the conversion of absorbed into emitted light; see eq 1.5,6 Dye performance is typically assessed using the brightness (ε(λex) × Φf) or the size of Φf7 and, for nanocrystalline chromophores like quantum dots, Φf presents a powerful tool to assess their quality.8,9
Φf =
Nem(λ) Nabs(λ)
(1)
Over the past decades, considerable efforts have been dedicated to develop reliable relative (standard needed) and absolute (standard-free) methods for the determination of Φf.6,10−21 This included optical methods as well as photothermal methods like photoacoustic spectroscopy and thermal lensing that determine this quantity indirectly from the measurement of dissipated heat.11 Commonly, Φf is obtained optically comparing the absorption-weighted integral fluorescence intensities of a sample and a standard of known Φf.22 Main factors governing the accuracy of these relative optical © 2011 American Chemical Society
Received: August 31, 2011 Accepted: December 22, 2011 Published: December 22, 2011 1345
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circumventing standard-related uncertainties8,12,35−37 Although such setups are more and more available, even calibrated, proper consideration of detector nonlinearities and reabsorption effects still present major challenges.22 The increasing importance of reliable fluorescence measurements caused us to assess methods, equipment, and standards for the measurement of Φf of application-relevant chromophores in the visible (vis) region and derive achievable uncertainties.10,22,24,38,39 These results, including the limitations of commercialized equipment,22,40 encouraged us to develop a new integrating sphere setup for the spectrally resolved measurements of absolute fluorescence quantum yields of transparent and scattering samples in the wavelength region of 400 to 1000 nm with measurement uncertainties ≤5% (for Φf values ≥10%). Other requirements included the suitability for direct and indirect sample illumination18 and the possibility to adjust the size of the excitation light beam to control the size of the illuminated sample volume. Here, we present the design of our integrating sphere setup and the calibration and validation strategies used including an uncertainty budget for the measurement of absolute fluorescence quantum yields of transparent dye solutions in the vis and NIR. As a first step to reliable Φf measurements in the long wavelength region, we assessed the potential of three commercialized red dyes as potential quantum yield standards for the spectral region of 600 to 1000 nm and present Φf values of common bioanalytically relevant vis and NIR emitters and an optical probe in mouse serum recently used for tumor imaging.
solution; Ca(II)- and Mg(II)-free without phenol red; pH = 7.8) used for ICG was obtained from PAA Laboratories (Pasching, Austria). Tris buffer (pH = 7.4) was prepared from tris-(hydroxymethyl)aminomethane and hydrochloric acid from Merck. Water equals always bidistilled water (pH ca. 7.0). Bovine serum albumin (BSA; fraction V) and silica particles were purchased from Merck. The mouse serum was selfextracted. All solvents were controlled for the presence of fluorescent impurities measuring blank spectra of the solvent for typical excitation wavelengths. Instrumentation. Relative Measurement of Φf. Absorption spectra were recorded on a calibrated Cary 5000 spectrometer. Fluorescence spectra were measured with a calibrated Spectronics Instruments 8100 with Glan Thompson polarizers in the excitation and the emission channel(s) set to 0° and 54.7°.10,23,44 All fluorescence emission and excitation spectra shown are blank-corrected and corrected for instrument-specific effects (see Supporting Information)23,25,45 Relative Φf values using a chain of Φf transfer standards consisting of n dyes with R101 acting as gold standard40 were calculated according to the formula of Demas and Crosby6 (see Supporting Information, eq 1S). Absolute Measurement of Φf. Absolute Φf values were determined with the newly built integrating sphere setup detailed in the Results and Discussion section. Additionally, absolute Φf values of selected dyes were measured with an integrating sphere setup C9920-02 from Hamamatsu Photonics.22 Measurement Conditions. All absorption and fluorescence measurements were performed with air-saturated freshly prepared dye solutions46 at T = (25 ± 1)°C using 10 mm × 10 mm as well as 4 mm × 10 mm quartz cuvettes from Hellma GmbH. For measurements with the commercial integrating sphere setup, 10 mm × 10 mm long-necked quartz cuvettes from Hamamatsu Photonics K.K. were employed, always filled with 3.5 mL of solvent or dye solution.22 For instrument calibration, at all times, expanded uncertainties (k = 2, confidence interval of 95%) are given. For all wavelengthdependent quantities (see Supporting Information, Table 3S), we used the relative standard deviation of the mean value, averaged over the relevant wavelength region. Dye Purity. HPLC analysis of the dyes detailed in the Supporting Information (Tables 4S and 5S) yielded the following purities: R101, 95.5% (525 nm) and 97.4% (565 nm); R6G, > 98.5% (480 nm, 530 nm); HITCI, 97.9% (760 nm); OXA1, 98.2% (665 nm); CRCY, 98.6% (725 nm); IR125, 99.1% (800 nm); and ICG, 98.8% (800 nm). The purity of QS of at least ≥98% follows from the NIST certificate and certification report,41 and the purity of the BAM spectral standards is at least ≥98.5%.23,42 All dyes were used without further purification. Safety Considerations. Proper safety procedures for handling, storage, and disposal of the organic dyes should be observed.
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MATERIALS AND INSTRUMENTATION Materials. Emission Standards. Quinine sulfate dihydrate (QS; equaling SRM 936a) was obtained from the National Institute of Standards and Technology (NIST).41 The spectral fluorescence standards F004 and F005 are from BAM and Sigma Aldrich.23,42 F007 is a new NIR emission standard currently evaluated by BAM. 4-(dicyanomethylene)-2-methyl6-(p-dimethylaminostyryl)-4H-pyran (DCM) was purchased from Lambda Physics (batch number 029401). All dyes were of the highest purity commercially available. Quantum Yield Standards. Rhodamine 101 (R101, batch number 019502), rhodamine 6G (R6G; batch number 119202), and the NIR dyes oxazine 1 (OXA1; batch number 090214), HITCI (batch number 029006), and IR 125 (batch number 10970) were obtained from Lambda Physics (see Supporting Information, Table 1S, for chemical structures). NIR Dyes, Labels, and Probes. Indocyanine green (ICG; 335601) was purchased from Pulsion Medical Systems, cryptocyanine (CRCY; batch 13715JZ) from Sigma Aldrich, Cy5, Cy5.5, and Cy7 from GE Healthcare Europe (batch numbers 392929, 398887 (Cy5.5 mono NHS ester, hydrolyzed; used for the preparation of RGD-Cy5.5), and 392393), Atto 740 from ATTO-Tec (batch number 131450324506P10), Atto 590 and Atto 680 from Fluka (batch numbers 70425 and 94875), and Alexa 647 and Alexa 750 from Invitrogen (batch numbers 822686 and 866762). The chemical structures of selected dyes are summarized in the Supporting Information (Table 2S). The synthesis of the new optical probe RGD-Cy5.5 was previously described.43 Solvents. The solvents used, i.e., ethanol for R101, R6G, OXA1, HITCI, CRCY, F004, F005, and F007, methanol for DCM, and dimethylsulfoxide (DMSO) for IR 125 were of spectroscopic grade and purchased from Sigma Aldrich and Merck. Phosphate buffer solution (PBS; Hank's balanced salt
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RESULTS AND DISCUSSION Design, Characterization, andHank Validation of the Integrating Sphere Setup. Instrument Design. For spectrally resolved measurements of absolute quantum yields in the wavelength region from ca. 400 to 1000 nm with versatile excitation and optimum spectral resolution, we used a 450 W xenon lamp, coupled to a single monochromator as excitation channel and an integrating sphere with a diameter of ca. 15 cm, 1346
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coated with Spectraflect (Labsphere; sphere reflectivity of about 97% in the vis/NIR), as sample compartment (Figure 1). The
fluctuations, a Peltier-cooled reference detector (customdesigned silicon trap detector)47 is implemented into the setup which is triggered with the shutter pulse of the CCD. To realize multiple measurement configurations, the integrating sphere was equipped with several ports for the coupling of light into the sphere and for sample mounting. For the determination of the absolute Φf with direct and indirect sample illumination, the sample is center-mounted inside the integrating sphere using a Spectraflect-coated cuvette holder positioned with a HeNe laser. The excitation light is focused with two off-axis parabolic mirrors into the middle (sample position) of the integrating sphere, thereby imaging the exit slit of the excitation monochromator onto the middle of the cuvette. To enable measurements of small sample volumes, the exit slit of the excitation monochromator can be changed in width and height, thus allowing the adjustment of the illuminated volume. The radiant power entering the integrating sphere is controlled by an aperture in front of the integrating sphere. Instrument Characterization. Prerequisites for accurate measurements of absolute Φf are the reliable determination of the wavelength accuracy of the excitation and detection channel(s), the linearity of the detection channel, the long-term stability of the radiant power of the excitation light source reaching the sample, and the spectral responsivity of the integrating sphere-spectrograph-CCD ensemble. Wavelength Accuracy. Control of the wavelength accuracy of the emission monochromator with a low pressure mercury/ argon discharge lamp25,26 (see Supporting Information, Figure 1S) reveals maximum deviations
