Ute Resch-Genger

Quantum dots versus organic dyes as fluorescent labels

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.

Relative and absolute determination of fluorescence quantum yields of transparent samples

Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ∼350–950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5–10% can be achieved within 2 h.

Near-infrared dyes for high technology applications

Preface. Advances in Methodology. Recent Developments in Fluorescence Spectroscopy: Three-Photon Excitation, Two-Color Two-Photon Excitation, Light Quenching and Development of Long-Lifetime Probes for Biophysics and Clinical Chemistry J.R. Lakowicz, et al. Near-Infrared Fluorescence Instrumentation for DNA Analysis L. Middendorf, et al. Highlights in Biochemistry and Biophysics. Ultrasensitive Detection and Identification of Biomolecules with Diode Lasers - from DYES to DNA M. Sauer, et al. Incorporation of Dye in Resting and Stimulated Leukocytes D. Frackowiak, et al. Application of Red Fluorescent Probes for the Measurement of Individual Cell Cytoplasmic pH Values J. Slavik. Molecular Probes Based on Cyanine Dyes for Nucleic Acid Research T.G. Deligeorgiev. New NIR Dyes: Synthesis, Spectral Properties and Applications in DNA Analyses N. Narayanan, et al. Frontiers in Analytical Chemistry and Sensor Technology. Luminescent Probes for NIR Sensing Applications E. Terpetschnig, O.S. Wolfbeis. NIR Fluorophores in Practical Analytical Chemistry A.R. Swamy, et al. Long Wavelength Emitting Fluorescence Probes for Metal Ions K. Rurack, et al. Advances in High Technology Applications. NIR Dyes for Information Recording, from Origin to Update M. Matsuoka. Tuning of Color by Different Alignment of Dye Molecules L. Dahne, E. Biller. Spectral Sensitization of Silver Halides in NIR Region B.I. Shapiro. Optical Properties and Applications of Near Infrared Dyes in Polymeric Media O.V. Przhonska. Insolubilisation and Fluorescence, Induced by Laser Diode Irradiation of IR-Dyes Embedded in Polymer Films. Thermally Induced Latex Coalescence and Acid Generation C.D. Catry, et al. NIRPhotosensitizers in Photodynamic Therapy E.A. Lukyanets. New Chromophores. Molecular Engineering of NIR Dyes S. Daehne, M.L. Dekhtyar. New NIR Dyes Based on the Cyclopentadienylium Chromophore and Related Compounds R. Gompper, et al. New Cyanine Dyes Absorbing in the NIR Region A.I. Tolmachev, et al. Near-Infrared Cyanine Dyes: A New Approach to an Old Problem Yu.L. Bricks, N.N. Romanov. Adventures in Search of New Dyes Absorbing in the Red or Near Infrared Region H. Hartmann. Subject Index.

Targeted luminescent near-infrared polymer-nanoprobes for in vivo imaging of tumor hypoxia.

Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.

Traceability in Fluorometry: Part II. Spectral Fluorescence Standards

The need for the traceable characterization of fluorescence instruments is emphasized from a chemist’s 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.

Image-guided, targeted and triggered drug delivery to tumors using polymer-based microbubbles

Microbubbles (MB) are routinely used contrast agents for functional and molecular ultrasound (US) imaging. In addition, they have been attracting more and more attention for drug delivery purposes, enabling e.g. US-mediated drug delivery across biological barriers and US-induced triggered drug release from the MB shell. The vast majority of efforts in this regard have thus far focused on phospholipid-based soft-shell MB, which are suboptimal for stably incorporating large amounts of drug molecules because of their relatively thin shell. Using poly(butyl cyanoacrylate) (PBCA)-based hard-shell MB, we show here that both hydrophilic (Rhodamine-B) and hydrophobic (Coumarin-6) model drugs can be efficiently and stably entrapped within the ~50 nm shell of PBCA MB. In addition, we demonstrate that model drug loading does not negatively affect the acoustic properties of the MB, and that functionalizing the surface of fluorophore-loaded MB with anti-VEGFR2 antibodies enables image-guided and targeted model drug delivery to tumor blood vessels. Finally, we show both in vitro and in vivo that disintegrating VEGFR2-targeted MB with high-mechanical index US pulses leads to high levels of model drug release. Consequently, these findings indicate that polymer-based MB are highly suitable systems for image-guided, targeted and triggered drug delivery to tumors and tumor blood vessels.

An in vitro characterization study of new near infrared dyes for molecular imaging

The spectroscopic properties, stability, and cytotoxicity of series of cyanine labels, the dyes DY-681, DY-731, DY-751, and DY-776, were studied to identify new tools for in vivo fluorescence imaging and to find substitutes for DY-676 recently used by us as fluorescent label in a target-specific probe directed against carcinoembryonic antigen (CEA). This probe enables the selective monitoring of CEA-expressing tumor cells in mice, yet displays only a low fluorescence quantum yield and thus, a non-optimum sensitivity. All the DY dyes revealed enhanced fluorescence quantum yields, a superior stability, and a lower cytotoxicity in comparison to clinically approved indocyanine green (ICG). With DY-681 and far-red excitable DY-731 and DY-751, we identified three dyes with improved properties compared to DY-676 and ICG.

Fluorescent anion receptors with iminoylthiourea binding sites—selective hydrogen bond mediated recognition of CO32−, HCO3− and HPO42−

Abstract A number of structurally simple, fluorescent sensor molecules based on the iminoylthiourea/1,2,4-thiadiazole unit are presented, which display extraordinarily strong fluorescence enhancement selectively upon complexation of HCO 3 − , CO 3 2− and HPO 4 2− .

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

Suitable Labels for Molecular Imaging – Influence of Dye Structure and Hydrophilicity on the Spectroscopic Properties of IgG Conjugates

Aiming at the design of highly brilliant NIR emissive optical probes, e.g., for in vivo near-infrared fluorescence imaging (NIRF), we studied the absorption and fluorescence properties of the asymmetric cyanines Dy678, Dy681, Dy682, and Dy676 conjugated to the model antibody IgG. The ultimate goal was here to derive general structure-property relationships for suitable NIR fluorescent labels. These Dy dyes that spectrally match Cy5 and Cy5.5, respectively, were chosen to differ in chromophore structure, i.e., in the substitution pattern of the benzopyrylium end group and in the number of sulfonic acid groups. Spectroscopic studies of the free and IgG-bound fluorophores revealed a dependence of the obtained dye-to-protein ratios on dye hydrophilicity and control of the fluorescence quantum yields (Φ(f)) of the IgG conjugates by the interplay of different fluorescence reduction pathways like dye aggregation and fluorescence resonance energy transfer (FRET). Based upon aggregation studies with these dyes, the amount of dye dimers in the IgG conjugates was determined pointing to dye hydrophilicity as major parameter controlling aggregation. To gain further insight into the exact mechanism of dye dimerization at the protein, labeling experiments at different reaction conditions but constant dye-to-protein ratios in the reaction solution were performed. With Dy682 that displays a Φ(f) of 0.20 in PBS and 0.10 for moderate dye-to-protein ratio of 2.5, a low aggregation tendency, and a superior reactivity in IgG labeling, we identified a promising diagnostic tool for the design of NIR fluorescent probes and protein conjugates.