Jutta Pauli

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.

Backbone and Side-Chain 13C and 15N Signal Assignments of the α-Spectrin SH3 Domain by Magic Angle Spinning Solid-State NMR at 17.6 Tesla

The backbone and side‐chain 13C and 15N signals of a solid 62‐residue (u‐13C,15N)‐labelled protein containing the α‐spectrin SH3 domain were assigned by two‐dimensional (2D) magic angle spinning (MAS) 15N–13C and 13C–13C dipolar correlation spectroscopy at 17.6 T. The side‐chain signal sets of the individual amino acids were identified by 2D 13C–13C proton‐driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton–nitrogen and a nitrogen–carbon cross‐polarisation step followed by a carbon–carbon homonuclear transfer unit. Interresidue correlations leading to sequence‐specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7–61, while the signals of the N‐ and C‐terminal residues 1–6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor‐bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three‐dimensional versions of the applied or analogous techniques.

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.

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.

Integrating Sphere Setup for the Traceable Measurement of Absolute Photoluminescence Quantum Yields in the Near Infrared

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 (Φ(f)) and brightness. The lack of well established Φ(f) standards for the NIR region in conjunction with the need for accurate Φ(f) 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 Φ(f) in the visible and NIR. To provide the basis for better measurements of Φ(f) in the spectral window from ca. 600 to 1000 nm used, e.g., for optical imaging, the absolute Φ(f) of a set of NIR chromophores covering this spectral region are measured and compared to relative values obtained using rhodamine 101 as Φ(f) standard. Additionally, the absolute Φ(f) 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.

Evaluation of a Commercial Integrating Sphere Setup for the Determination of Absolute Photoluminescence Quantum Yields of Dilute Dye Solutions

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 Vivo Near-infrared Fluorescence Imaging of Carcinoembryonic Antigen–expressing Tumor Cells in Mice

PURPOSE To prospectively depict carcinoembryonic antigen (CEA)-expressing tumors in mice with a high-affinity probe consisting of a near-infrared (NIR) fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab. MATERIALS AND METHODS This study was approved by the regional animal committee. By coupling a NIR fluorescent (NIRF) cyanine dye (DY-676) to a specific antibody fragment directed against CEA (arcitumomab) and a nonspecific IgG Fab fragment, a bio-optical high-affinity fluorescent probe (anti-CEA-DY-676) and a low-affinity fluorescent probe (FabIgG-DY-676) were designed. The dye-to-protein ratios were determined, and both probes were tested for NIRF imaging in vitro on CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells by using a bio-optical NIR small-animal imager. In vivo data of xenografted LS-174T and A-375 tumors in mice (n = 10) were recorded and statistically analyzed (Student t test). RESULTS The dye-to-protein ratios were determined as 3.0-3.5 for both probes. In vitro experiments revealed the specific binding of the anti-CEA-DY-676 probe on CEA-expressing cells as compared with CEA-nonexpressing cells; the FabIgG-DY-676 probe showed a markedly lower binding affinity to cells. In vivo LS-174T tumors xenografted in all mice could be significantly distinguished from A-375 tumors with application of the anti-CEA-DY-676 but not with that of the FabIgG-DY-676 at different times (2-24 hours, P < .005) after intravenous injection of the probes. Semiquantitative analysis revealed maximal fluorescence signals of anti-CEA-DY-676 to CEA-expressing tumors about 8 hours after injection. CONCLUSION Findings of this study indicate the potential use of the high-affinity probe anti-CEA-DY-676 for specific NIRF imaging in in vivo tumor diagnosis.

Novel Fluorophores as Building Blocks for Optical Probes for In Vivo Near Infrared Fluorescence (NIRF) Imaging

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.

Assignment of the Nonexchanging Protons of theα-Spectrin SH3 Domain by Two- and Three- Dimensional1H-13C Solid-State Magic-Angle Spinning NMR and Comparison of Solution and Solid-State Proton Chemical Shifts

The assignment of nonexchanging protons of a small microcrystalline protein, the α‐spectrin SH3 domain (7.2 kDa, 62 residues), was achieved by means of three‐dimensional (3D) heteronuclear (1H–13C–13C) magic‐angle spinning (MAS) NMR dipolar correlation spectroscopy. With the favorable combination of a high B0‐field, a moderately high spinning frequency, and frequency‐switched Lee‐Goldburg irradiation applied during 1H evolution, a proton linewidth ≤0.5 ppm at 17.6 Tesla was achieved for the particular protein preparation used. A comparison of the solid‐state 1H chemical shifts with the shifts found in solution shows a remarkable similarity, which reflects the identical protein structures in solution and in the solid. Significant differences between the MAS solid‐ and liquid‐state 1H chemical shifts are only observed for residues that are located at the surface of the protein and that exhibit contacts between different SH3 molecules. In two cases, aromatic residues of neighboring SH3 molecules induce pronounced upfield ring‐current shifts for protons in the contact area.

New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging.

The rational design of bright optical probes and dye-biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M(-1) cm(-1) and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.