TY - JOUR A1 - Würth, Christian A1 - Pauli, Jutta A1 - Lochmann, Cornelia A1 - Spieles, Monika A1 - Resch-Genger, Ute T1 - Integrating sphere setup for the traceable measurement of absolute photoluminescence quantum yields in the near infrared N2 - 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. KW - Fluorescence KW - Photoluminescence KW - Quantum yield KW - Dye KW - Uncertainty KW - Absolute quantum yield KW - Method comparison KW - Integrating sphere KW - NIR KW - Standards KW - Quantum yield standards PY - 2012 DO - https://doi.org/10.1021/ac2021954 SN - 0003-2700 SN - 1520-6882 VL - 84 IS - 3 SP - 1345 EP - 1352 PB - American Chemical Society CY - Washington, DC AN - OPUS4-25504 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Behnke, Thomas A1 - Würth, Christian A1 - Laux, Eva-Maria A1 - Hoffmann, Katrin A1 - Resch-Genger, Ute T1 - Simple strategies towards bright polymer particles via one-step staining procedures N2 - In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles. KW - Fluorescence KW - Polystyrene KW - Particles KW - Encapsulation KW - Quantum yield KW - Zeta potential KW - Method KW - Label KW - Particle KW - Polymer KW - Absolute fluorescence quantum yield KW - Fluorophore KW - Dye content KW - Surface groups KW - Size KW - Brightness PY - 2012 DO - https://doi.org/10.1016/j.dyepig.2012.01.021 SN - 0143-7208 SN - 1873-3743 VL - 94 IS - 2 SP - 247 EP - 257 PB - Elsevier Ltd. CY - Kidlington AN - OPUS4-25481 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Resch-Genger, Ute A1 - Behnke, Thomas A1 - Brehm, Robert A1 - Grabolle, Markus A1 - Hennig, Andreas A1 - Hoffmann, Angelika A1 - Hoffmann, Katrin A1 - Linck, Lena A1 - Lochmann, Cornelia A1 - Pauli, Jutta A1 - Spieles, Monika A1 - Würth, Christian T1 - Funktionelle Chromophor-Systeme, innovative Validierungskonzepte und rückführbare Standards für die fluoreszenzbasierte multiparametrische Bioanalytik N2 - 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. T2 - 5. Senftenberger Innovationsforum Multiparameteranalytik CY - Senftenberg, Deutschland DA - 10.03.2011 KW - Multiparametric KW - Multiplexing KW - Fluorescence KW - Nanoparticles KW - NIR dyes KW - Surface analysis KW - Quantum yield KW - Quantum dot KW - Lifetime PY - 2011 SP - 86 EP - 108 CY - Senftenberg AN - OPUS4-23635 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Würth, Christian A1 - Grabolle, Markus A1 - Pauli, Jutta A1 - Spieles, Monika A1 - Resch-Genger, Ute T1 - Comparison of methods and achievable uncertainties for the relative and absolute measurement of photoluminescence quantum yields N2 - 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. KW - Fluorescence KW - Photoluminescence KW - Quantum yield KW - Dye KW - Uncertainty KW - Absolute quantum yield KW - Method comparison KW - Integrating sphere PY - 2011 DO - https://doi.org/10.1021/ac2000303 SN - 0003-2700 SN - 1520-6882 VL - 83 IS - 9 SP - 3431 EP - 3439 PB - American Chemical Society CY - Washington, DC AN - OPUS4-23632 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Würth, Christian A1 - Lochmann, Cornelia A1 - Spieles, Monika A1 - Pauli, Jutta A1 - Hoffmann, Katrin A1 - Schüttrigkeit, T. A1 - Franzl, T. A1 - Resch-Genger, Ute T1 - Evaluation of a commercial integrating sphere setup for the determination of absolute photoluminescence quantum yields of dilute dye solutions N2 - 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. KW - Lifetime KW - Fluorescence KW - Luminescence KW - Quantum yield KW - Quantum efficiency KW - Integrating sphere KW - Reabsorption KW - Rhodamine 101 KW - Quinine sulfate dihydrate KW - Method KW - Photoluminescence KW - Standard KW - Emission KW - Spectral correction KW - Excitation KW - Anisotropy PY - 2010 DO - https://doi.org/10.1366/000370210791666390 SN - 0003-7028 SN - 1943-3530 VL - 64 IS - 7 SP - 733 EP - 741 PB - Society for Applied Spectroscopy CY - Frederick, Md. AN - OPUS4-22089 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pauli, Jutta A1 - Brehm, Robert A1 - Spieles, Monika A1 - Kaiser, W.A. A1 - Hilger, I. A1 - Resch-Genger, Ute T1 - Novel fluorophores as building blocks for optical probes for in vivo near infrared fluorescence (NIRF) imaging N2 - Aiming at the identification of new fluorescent reporters for targeted optical probes, we assessed the application-relevant features of a novel asymmetric cyanine, DY-681, in comparison to the only clinically approved dye indocyanine green (ICG), the golden imaging standard Cy5.5, and the asymmetric cyanine DY-676 successfully exploited by us for the design of different contrast agents. This comparison included the analysis of the spectroscopic properties of the free fluorophores and their thermal stability in aqueous solution as well as their cytotoxic potential. In addition, the absorption and emission features of IgG-conjugated DY-681 were examined. The trimethine DY-681 exhibited spectral features closely resembling that of the pentamethine Cy5.5. Its high thermal stability in phosphate buffer saline (PBS) solution in conjunction with its low cytotoxicity, reaching similar values as determined for Cy5.5 and DY-676, renders this dye more attractive as ICG and, due to its improved fluorescence quantum yield in PBS, also superior to DY-676. Although in PBS, Cy5.5 was still more fluorescent, the fluorescence quantum yields (Φf) of DY-681 and Cy5.5 in PBS containing 5 mass-% bovine serum albumin (BSA) were comparable. Labeling experiments with DY-681 and the model antibody IgG revealed promisingly high Φf values of the bioconjugated dye. KW - Fluorescence KW - Cyanine dye KW - Cytotoxicity KW - Stability KW - In vivo fluorescence imaging KW - Quantum yield KW - Contrast agent KW - Optical probe PY - 2010 DO - https://doi.org/10.1007/s10895-010-0603-7 SN - 1053-0509 SN - 1573-4994 VL - 20 IS - 3 SP - 681 EP - 693 PB - Plenum Publ. Corp. CY - New York, NY AN - OPUS4-21401 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - DeRose, P.C. A1 - Resch-Genger, Ute T1 - Recommendations for fluorescence instrument qualification: the new ASTM standard guide N2 - Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements. KW - Fluorescence KW - Method KW - Quantum yield KW - Photoluminescence KW - Standard KW - Emission KW - Spectral correction KW - Excitation KW - Anisotropy KW - Recommendation KW - Instrument qualification PY - 2010 DO - https://doi.org/10.1021/ac902507p SN - 0003-2700 SN - 1520-6882 VL - 82 IS - 5 SP - 2129 EP - 2133 PB - American Chemical Society CY - Washington, DC AN - OPUS4-21066 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Pfeifer, Dietmar ED - Chris D. Geddes, T1 - Simple calibration and validation standards for fluorometry KW - Fluorescence KW - Standard KW - Spectral correction KW - Emission KW - Glass KW - Quantum yield KW - Fluorescence intensity KW - Quality assurance PY - 2009 SN - 978-0-387-88721-0 DO - https://doi.org/10.1007/978-0-387-88722-7_1 SN - 1573-8086 N1 - Serientitel: Reviews in Fluorescence – Series title: Reviews in Fluorescence VL - 4 SP - 1 EP - 31 PB - Springer Science + Business Media AN - OPUS4-19830 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Resch-Genger, Ute A1 - Grabolle, Markus A1 - Cavaliere-Jaricot, S. A1 - Nitschke, R. A1 - Nann, T. T1 - Quantum dots versus organic dyes as fluorescent labels N2 - Suitable labels are at the core of luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)—inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry—as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels. KW - Fluorescence KW - Label KW - Quantum dot KW - Dye KW - Bioanalysis KW - Quantum yield KW - Multiplexing KW - Lifetime KW - Assay KW - Life sciences KW - FRET KW - Fluorescent reporter PY - 2008 DO - https://doi.org/10.1038/NMETH.1248 SN - 1548-7091 SN - 1548-7105 VL - 5 IS - 9 SP - 763 EP - 775 PB - Nature Publishing Group CY - New York, NY, USA AN - OPUS4-18992 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Grabolle, Markus A1 - Ziegler, J. A1 - Merkulov, A. A1 - Nann, T. A1 - Resch-Genger, Ute T1 - Stability and Fluorescence Quantum Yield of CdSe-ZnS Quantum Dots - Influence of the Thickness of the ZnS Shell N2 - We investigated the correlation between the thickness of the ZnS shell of CdSe–ZnS quantum dots (QDs), the stability of the particles, and the fluorescence quantum yield. As a measure for stability, a new shell quality test was developed. This test is based on the reaction of the QDs with photochemically formed thiophenol radicals and communicates an imperfect ZnS shell by a rapid and complete loss of fluorescence. The quantum yield increases from less than 5% for unshelled CdSe up to 50%, with an increase in ZnS shell thickness up to 0.6–0.8 nm. At the same time, the particles become significantly more stable, as revealed by the shell test. KW - Quantum dot KW - Nanocrystal KW - Semiconductor KW - Fluorescence KW - Quantum yield KW - CdSe KW - CdSe-ZnS KW - Shell KW - ZnS shell KW - Stability KW - Stability test PY - 2008 DO - https://doi.org/10.1196/annals.1430.021 SN - 0077-8923 SN - 1749-6632 SN - 0094-8500 VL - 1130 SP - 235 EP - 241 PB - New York Academy of Sciences CY - New York, NY AN - OPUS4-17743 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -