The need for the traceable characterization of fluorescence instruments is emphasized from a chemists point of view, focusing on spectral fluorescence standards for the determination of the wavelength- and polarization-dependent relative spectral responsivity and relative spectral irradiance of fluorescence measuring systems, respectively. In a first step, major sources of error of fluorescence measurements and instrument calibration are revealed to underline the importance of this issue and to illustrate advantages and disadvantages of physical and chemical transfer standards for generation of spectral correction curves. Secondly, examples for sets of traceable chemical emission and excitation standards are shown that cover a broad spectral region and simple procedures for the determination of corrected emission spectra with acceptable uncertainties are presented. With proper consideration of the respective measurement principle and geometry, these dye-based characterization procedures can be not only applied to spectrofluorometers but also to other types of fluorescence measuring systems and even to Raman spectrometers.
Four novel borondipyrromethene (BDP) and -diindomethene (BDI) dyes with one or two (dimethylamino)styryl extensions at the chromophore were synthesized and spectroscopically investigated. An X-ray crystal structure shows that the extended auxochrome is virtually planar. All dyes thus display intense red/near infrared (NIR) absorption and emission. The (dimethylamino)styryl group induces a charge-transfer character that entails bright solvatochromic fluorescence, which is only quenched with increasing solvent polarity according to the energy-gap law. The dye with an additional dimethylanilino group at the meso position of BDP shows a remarkable switching of lipophilicity by protonation. Two dyes with an 8-hydroxyquinoline ligand at the meso position display quenched emission in the presence of Hg2+ or Al3+ owing to electron transfer from the excited BDP to the complexed receptor. The BDI dye presents a pH indicator with bright fluorescence and extremely low fluorescence anisotropy.
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.
The X-ray crystallographic, optical spectroscopic, and electrochemical properties of a newly synthesized class of boron-diindomethene (BDI) dyes and their tetrahydrobicyclo precursors (bc-BDP) are presented. The BDI chromophore was designed to show intensive absorption and strong fluorescence in an applicationary advantageous spectral range. Its modular architecture permits fusion of a second subunit, for example, a receptor moiety to the dye's core to yield directly linked yet perpendicularly prearranged composite systems. The synthesis was developed to allow facile tuning of the chromophore platform and to thus adjust its redox properties. X-ray analysis revealed a pronounced planarity of the chromophore in the case of the BDIs, which led to a remarkable close packing in the crystal of the simplest derivative. On the other hand, deviation from planarity was found for the diester-substituted bc-BDP benzocrown that exhibits a butterfly-like conformation in the crystal. Both families of dyes show charge- or electron-transfer-type fluorescence-quenching characteristics in polar solvents when equipped with a strong donor in the meso-position of the core. These processes can be utilized for signaling purposes if an appropriate receptor is introduced. Further modification of the chromophore can invoke such a guest-responsive intramolecular quenching process, also for receptor groups of low electron density, for example, benzocrowns. In addition to the design of various prototype molecules, a promising fluoroionophore for Na+ was obtained that absorbs and emits in the 650 nm region and shows a strong fluorescence enhancement upon analyte binding. Furthermore, investigation of the remarkable solvatokinetic fluorescence properties of the butterfly-like bc-BDP derivatives suggested that a second intrinsic nonradiative deactivation channel can play a role in the photophysics of boron-dipyrromethene dyes.
The commercial availability of stand-alone setups for the determination of absolute photoluminescence quantum yields (φf) in conjunction with the increasing use of integrating sphere accessories for spectrofluorometers is expected to have a considerable influence not only on the characterization of chromophore systems for use in optical and opto-electronic devices, but also on the determination of this key parameter for (bio)analytically relevant dyes and functional luminophores. Despite the huge potential of systems measuring absolute φf values and the renewed interest in dependable data, evaluated protocols for even the most elementary case, the determination of the fluorescence quantum yield of transparent dilute solutions of small organic dyes with integrating sphere methods, are still missing. This encouraged us to evaluate the performance and sources of uncertainty of a simple commercial integrating sphere setup with dilute solutions of two of the best characterized fluorescence quantum yield standards, quinine sulfate dihydrate and rhodamine 101, strongly differing in spectral overlap between absorption and emission. Special attention is dedicated to illustrate common pitfalls of this approach, thereby deriving simple procedures to minimize measurement uncertainties and improve the comparability of data for the broad community of users of fluorescence techniques.
In the second part of this two-part series on the state-of-the-art comparability of corrected emission spectra, we have extended this assessment to the broader community of fluorescence spectroscopists by involving 12 field laboratories that were randomly selected on the basis of their fluorescence measuring equipment. These laboratories performed a reference material (RM)-based fluorometer calibration with commercially available spectral fluorescence standards following a standard operating procedure that involved routine measurement conditions and the data evaluation software LINKCORR developed and provided by the Federal Institute for Materials Research and Testing (BAM). This instrument-specific emission correction curve was subsequently used for the determination of the corrected emission spectra of three test dyes, X, QS, and Y, revealing an average accuracy of 6.8% for the corrected emission spectra. This compares well with the relative standard uncertainties of 4.2% for physical standard-based spectral corrections demonstrated in the first part of this study (previous paper in this issue) involving an international group of four expert laboratories. The excellent comparability of the measurements of the field laboratories also demonstrates the effectiveness of RM-based correction procedures.
The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods. The results, obtained for typical spectrofluorometric (0°/90° transmitting) and colorimetric (45°/0° front-face) measurement geometries, demonstrated a comparability of corrected emission spectra within a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based spectral correction when measurements and calibrations were performed under identical conditions. Moreover, the emission spectra of RMs F001 to F005, certified by BAM, Federal Institute for Materials Research and Testing, were confirmed. These RMs were subsequently used for the assessment of the comparability of RM-based corrected emission spectra of field laboratories using common commercial spectrofluorometers and routine measurement conditions in part 2 of this series (subsequent paper in this issue).
The photoluminescence quantum yield (Φf) that presents a direct measure for the efficiency of the conversion of absorbed photons into emitted photons is one of the spectroscopic key parameters of functional fluorophores. It determines the suitability of such materials for applications in, for example, (bio)analysis, biosensing, and fluorescence imaging as well as as active components in optical devices. The reborn interest in accurate Φf measurements in conjunction with the controversial reliability of reported Φf values of many common organic dyes encouraged us to compare two relative and one absolute fluorometric method for the determination of the fluorescence quantum yields of quinine sulfate dihydrate, coumarin 153, fluorescein, rhodamine 6G, and rhodamine 101. The relative methods include the use of a chain of Φf transfer standards consisting of several 'standard dye' versus 'reference dye' pairs linked to a golden Φf standard that covers the ultraviolet and visible spectral region, and the use of different excitation wavelengths for standard and sample, respectively. Based upon these measurements and the calibration of the instruments employed, complete uncertainty budgets for the resulting Φf values are derived for each method, thereby providing evaluated standard operation procedures for Φf measurements and, simultaneously, a set of assessed Φf standards.
Unter dem Motto „Innovation und Qualitätssicherung in der (Bio)Analytik“ werden in der Arbeitsgruppe Fluoreszenzspektroskopie der BAM, Bundesanstalt für Materialforschung und -prüfung, funktionelle Chromophor-Systeme, einfache Signalverstärkungs- und Multiplexingstrategien sowie innovative Validierungs- und rückführbare Standardisierungskonzepte für verschiedene fluorometrische Messgrößen und Methoden entwickelt. Im Mittelpunkt stehen dabei molekulare Fluorophore, Nanokristalle mit größenabhängigen optischen Eigenschaften (sogenannte Quantenpunkte, QDs) und fluoreszierende Partikel variabler Größe sowie Sonden und Sensormoleküle für neutrale und ionische Analyte und für die Charakterisierung von funktionellen Gruppen. Dabei erfolgen auch methodische Entwicklungen für die Fluoreszenzspektroskopie, die Fluoreszenzmikroskopie, die Milcrofluorometrie, die Sensorik und die Mikroarraytechnologie. Ziele sind u. a. das Design und die Untersuchung von multiplexfähigen selektiven und sensitiven Sonden für die Biomarkeranalytik, die Entwicklung von Methoden zur Charakterisierung der signalrelevanten Eigenschaften dieser Chromophor-Systeme und zur Charakterisierung von funktionellen Gruppen an Oberflächen und ihre Validierung sowie die Entwicklung und Bereitstellung von formatadaptierbaren, flexibel ersetzbaren
Standards für die fluoreszenzbasierte Multiparameteranalytik.