TY - JOUR A1 - Hoffmann, Katrin A1 - Behnke, Thomas A1 - Grabolle, Markus A1 - Resch-Genger, Ute T1 - Nanoparticle-encapsulated vis- and NIR-emissive fluorophores with different fluorescence decay kinetics for lifetime multiplexing N2 - Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding. KW - Fluorescent label KW - Multiplexing KW - Optical KW - encoding KW - Lifetime multiplexing KW - Fluorescence lifetime imaging FLIM KW - Nanoparticles PY - 2014 DO - https://doi.org/10.1007/s00216-013-7597-3 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 14 SP - 3315 EP - 3322 PB - Springer CY - Berlin AN - OPUS4-30077 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Grabolle, Markus A1 - Pauli, Jutta A1 - Brehm, Robert A1 - Resch-Genger, Ute T1 - Structural control of dye-protein binding, aggregation and hydrophilicity in a series of asymmetric cyanines N2 - Aiming at the rational design and the identification of brilliant fluorescent reporters for targeted optical probes and fluorescence assays in biological matrices, we systematically assessed the correlation between dye–protein binding, dye aggregation, and dye hydrophilicity for bioanalytically relevant fluorescent labels. Here, we report on the influence of sulfonic acid groups on dye aggregation and dye–serum protein interactions exemplarily for a family of NIR-emissive cyanine dyes, the DY-67x fluorophores. For highly hydrophobic dyes like DY-675 and DY-676, which show a strong tendency for aggregation in phosphate buffer saline solution, the dye–protein binding constants determined spectroscopically using a 2-state binding model, which considers only protein-bound and unbound dye molecules, can be influenced by the dimerization of the unbound dyes. To consider and quantify this influence, we expanded this common photometric method to a 3-state model that accounts for the presence of dye aggregates in the binding studies. Our results can be exploited for the screening of fluorescent reporters, efficiently providing information on the size of dye–protein interactions and on maximally achievable fluorescence quantum yields in biological systems. KW - Cyanine dye KW - Dye–protein interaction KW - BSA KW - Binding constant KW - Hydrophilicity KW - Aggregation PY - 2014 DO - https://doi.org/10.1016/j.dyepig.2013.11.027 SN - 0143-7208 SN - 1873-3743 VL - 103 SP - 118 EP - 126 PB - Elsevier Ltd. CY - Kidlington AN - OPUS4-30054 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Resch-Genger, Ute A1 - Hatami, Soheil A1 - Würth, Christian A1 - Kaiser, Martin A1 - Leubner, S. A1 - Graponik, N. A1 - Eychmüller, A. T1 - Measurement of Absolute Fluorescence Quantum Yields in the Near-Infrared and Infrared Spectral Region T2 - SPIE Photonics West 2014 (international society for optics and photonics) CY - San Francisco, CA, USA DA - 2014-02-01 PY - 2014 AN - OPUS4-30199 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Resch-Genger, Ute A1 - Hatami, Soheil A1 - Kaiser, Martin A1 - Würth, Christian A1 - Leubner, S. A1 - Moser, Marko A1 - Schneider, Ralf A1 - Gaponik, N. A1 - Eychmüller, A. T1 - Optical Properties and Surface Chemistry of Quantum Dots, Polymeric and Upconversion Nanoparticles T2 - SPIE Photonics West 2014 (international society for optics and photonics) CY - San Francisco, CA, USA DA - 2014-02-01 PY - 2014 AN - OPUS4-30200 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Dietrich, Paul A1 - Hennig, Andreas A1 - Holzweber, Markus A1 - Thiele, T. A1 - Borcherding, H. A1 - Lippitz, Andreas A1 - Schedler, U. A1 - Resch-Genger, Ute A1 - Unger, Wolfgang T1 - Surface analytical study of poly(acrylic acid)-grafted microparticles (beads): characterization, chemical derivatization, and quantification of surface carboxyl groups N2 - We report a surface analytical study of poly(methyl methacrylate) (PMMA) microparticles (beads) with a grafted shell of poly(acrylic acid) (PAA) with thicknesses up to 4 nm using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy. These polymer microparticles were analyzed before and after reaction of the surface carboxyl (CO2H) groups with 2,2,2-trifluoroethylamine (TFEA) to gain a better understanding of methods with use of covalently bound probe molecules for surface group analysis. The results obtained with chemical derivatization XPS using TFEA are discussed in terms of surface quantification of reactive CO2H groups on these PAA-coated microparticles. A labeling yield of about 50% was found for TFEA-derivatized particles with amounts of surface-grafted CO2H groups of 99 µmol/g or more, which is consistent with predicted reaction yields for homogeneously dispersed PAA hydrogels. KW - Polymer microparticles KW - Poly(acrylic acid)-grafted microparticles KW - Beads KW - XPS KW - SEM KW - NEXAFS KW - Surface analysis KW - Fluorine labeling PY - 2014 DO - https://doi.org/10.1021/jp505519g SN - 1932-7447 SN - 1089-5639 VL - 118 IS - 35 SP - 20393 EP - 20404 PB - Soc. CY - Washington, DC AN - OPUS4-31326 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schoetz, U. A1 - Deliolanis, N.C. A1 - Ng, D. A1 - Pauli, Jutta A1 - Resch-Genger, Ute A1 - Kühn, E. A1 - Heuer, S. A1 - Beisker, W. A1 - Köster, R.W. A1 - Zitzelsberger, H. A1 - Caldwell, R.B. T1 - Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure N2 - With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model. KW - Fluorescent protein KW - Quantum yield PY - 2014 DO - https://doi.org/10.1371/journal.pone.0107069 SN - 1932-6203 VL - 9 IS - 9 SP - e107069-1 EP - e107069-12 PB - PubMed Central AN - OPUS4-31976 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Pauli, Jutta ED - Brahme, A. T1 - Signal-relevant properties of fluorescent labels and optical probes and their determination PY - 2014 SN - 978-0-444-53632-7 DO - https://doi.org/10.1016//B978-0-444-53632-7.00408-1 VL - 4 SP - Chapter 4.02, 15 EP - 26 PB - Elsevier CY - Amsterdam AN - OPUS4-31433 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Licha, K. A1 - Resch-Genger, Ute ED - Brahme, A. T1 - Fluorescent reporters and optical probes N2 - Organic fluorescent dyes are a fundamental component in biomedical research and diagnostic imaging. The major classes encompass polymethine dyes, xanthene dyes, 4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes, phenoxazines, and rare earth metal complexes. These compounds have been synthesized in manifold variations to optimize their photophysical properties and physicochemical behavior in physiological media and to enable conjugation to targeting molecules and nanocarriers. Furthermore, stimuli-responsive structural motifs were designed to apply these fluorophores as sensors for disease-related physiological and molecular conditions. This chapter is devoted to the chemical base of optical imaging agents, covering the relevant properties of fluorophores and the synthetic concepts toward intelligent optical imaging probes. KW - Chemistry KW - Fluorescent dye KW - Labeling KW - Near-infrared KW - Optical imaging KW - Sensor PY - 2014 SN - 978-0-444-53632-7 DO - https://doi.org/10.1016/B978-0-444-53632-7.00402-0 VL - 4 SP - Chapter 4.08, 85 EP - 109 PB - Elsevier CY - Amsterdam AN - OPUS4-31635 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Leubner, S. A1 - Schneider, Ralf A1 - Dubavik, A. A1 - Hatami, Soheil A1 - Gaponik, N. A1 - Resch-Genger, Ute A1 - Eychmüller, A. T1 - Influence of the stabilizing ligand on the quality, signal-relevant optical properties, and stability of near-infrared emitting Cd1-xHgxTe nanocrystals N2 - Bright and stable near-infrared (NIR) and infrared (IR) emitting chromophores are in high demand for applications in telecommunication, solar cells, security barcodes, and as fluorescent reporters in bioimaging studies. The best choice for wavelengths >750 nm are semiconductor nanocrystals, especially ternary or alloy nanocrystals like CdHgTe, which enable size and composition control of their optical properties. Here, we report on the influence of growth time and surface chemistry on the composition and optical properties of colloidal CdHgTe. Up to now, these are the only NIR and IR emissive quantum dots, which can be synthesized in high quality in water, using a simple one-pot reaction. For this study we utilized and compared three different thiol ligands, thioglycolic acid (TGA), 3-mercaptopropionic acid (MPA), and glutathione (GSH). Aiming at the rational design of bright NIR- and IR-emissive alloy materials, special emphasis was dedicated to a better understanding of the role of the surface ligand and adsorption–desorption equilibria on the photoluminescence quantum yield and stability. In this respect, dilution and protonation studies were performed. Our results show that with this simple synthetic procedure, strongly fluorescent CdHgTe colloids can be obtained with MPA as stabilizing ligand revealing quantum yields as high as 45% independent of particle concentration. PY - 2014 DO - https://doi.org/10.1039/c4tc00582a SN - 2050-7526 SN - 2050-7534 VL - 2 IS - 25 SP - 5011 EP - 5018 PB - Royal Soc. of Chemistry CY - Cambridge AN - OPUS4-31073 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Müller, M. A1 - Kaiser, Martin A1 - Stachowski, G.M. A1 - Resch-Genger, Ute A1 - Gaponik, N. A1 - Eychmüller, A. T1 - Photoluminescence quantum yield and matrix-induced luminescence enhancement of colloidal quantum dots embedded in ionic crystals N2 - The incorporation of colloidal quantum dots (QDs) into solid matrices, especially ionic salts, holds several advantages for industrial applications. Here, we demonstrated via absolute measurements of photoluminescence quantum yields (PL-QY) that the photoluminescence of aqueous CdTe QDs can be considerably increased upon incorporation into a salt matrix with a simple crystallization procedure. Enhancement factors of up to 2.8 and a PL-QY of 50 to 80%, both in NaCl crystals and incorporated in silicone matrices, were reached. The fact that the achievable PL enhancement factors depend strongly on PL-QY of the parent QDs can be described by the change of the dielectric surrounding and the passivation of the QD surface, modifying radiative and nonradiative rate constants. Time-resolved PL measurements revealed noncorrelating PL lifetimes and PL-QY, suggesting that weakly emissive QDs of the ensemble are more affected by the enhancement mechanism, thereby influencing PL-QY and PL lifetime in a different manner. PY - 2014 DO - https://doi.org/10.1021/cm5009043 SN - 0897-4756 SN - 1520-5002 VL - 26 IS - 10 SP - 3231 EP - 3237 PB - American Chemical Society CY - Washington, DC AN - OPUS4-31074 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -