Christian Würth

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.

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.

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.

Determination of the absolute fluorescence quantum yield of rhodamine 6G with optical and photoacoustic methods – Providing the basis for fluorescence quantum yield standards

To establish the methodical basis for the development and certification of fluorescence quantum yield standards, we determined the fluorescence quantum yield Φ(f) of rhodamine 6G (R6G) with two absolute methods with complementary measurement principles, here optical spectroscopy using an integrating sphere setup and pulsed laser photoacoustic spectroscopy (PAS). For the assessment of aggregation- and reabsorption-induced distortions of measured fluorescence quantum yields and procedures for the reliable consideration of such effects, this systematic comparison was performed in ethanol and in water employing different concentrations of R6G. In addition, the relative and absolute fluorescence quantum yields of these solutions were obtained with a calibrated spectrofluorometer and a commercialized integrating sphere setup. Based upon this systematic comparison, experimental advantages and systematic sources of variation were identified for both methods.

Encapsulation of Hydrophobic Dyes in Polystyrene Micro- and Nanoparticles via Swelling Procedures

Aiming at the derivation of a generalized procedure for the straightforward preparation of particles fluorescing in the visible and near-infrared (NIR) spectral region, different swelling procedures for the loading of the hydrophobic polarity-probe Nile Red into nano- and micrometer sized polystyrene particles were studied and compared with respect to the optical properties of the resulting particles. The effect of the amount of incorporated dye on the spectroscopic properties of the particles was investigated for differently sized beads with different surface chemistries, i.e., non-functionalized, amino-modified and PEG-grafted surfaces. Moreover, photostability and leaking studies were performed. The main criterion for the optimization of the dye loading procedures was a high and thermally and photochemically stable fluorescence output of the particles for the future application of these systems as fluorescent labels.

Scope and Limitations of Surface Functional Group Quantification Methods: Exploratory Study with Poly(acrylic acid)-Grafted Micro- and Nanoparticles

The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, (13)C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network.

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.

Target-specific nanoparticles containing a broad band emissive NIR dye for the sensitive detection and characterization of tumor development

Current optical probes including engineered nanoparticles (NPs) are constructed from near infrared (NIR)-emissive organic dyes with narrow absorption and emission bands and small Stokes shifts prone to aggregation-induced self-quenching. Here, we present the new asymmetric cyanine Itrybe with broad, almost environment-insensitive absorption and emission bands in the diagnostic window, offering a unique flexibility of the choice of excitation and detection wavelengths compared to common NIR dyes. This strongly emissive dye was spectroscopically studied in different solvents and encapsulated into differently sized (15, 25, 100 nm) amino-modified polystyrene NPs (PSNPs) via a one-step staining procedure. As proof-of-concept for its potential for pre-/clinical imaging applications, Itrybe-loaded NPs were surface-functionalized with polyethylene glycol (PEG) and the tumor-targeting antibody Herceptin and their binding specificity to the tumor-specific biomarker HER2 was systematically assessed. Itrybe-loaded NPs display strong fluorescence signals in vitro and in vivo and Herceptin-conjugated NPs bind specifically to HER2 as demonstrated in immunoassays as well as on tumor cells and sections from mouse tumor xenografts in vitro. This demonstrates that our design strategy exploiting broad band-absorbing and -emitting dyes yields versatile and bright NIR probes with a high potential for e.g. the sensitive detection and characterization of tumor development and progression.

Critical review of the determination of photoluminescence quantum yields of luminescent reporters.

AbstractA crucial variable for methodical performance evaluation and comparison of luminescent reporters is the photoluminescence quantum yield (Φpl). This quantity, defined as the number of emitted photons per number of absorbed photons, is the direct measure of the efficiency of the conversion of absorbed photons into emitted light for small organic dyes, fluorescent proteins, metal–ligand complexes, metal clusters, polymeric nanoparticles, and semiconductor and up-conversion nanocrystals. Φpl determines the sensitivity for the detection of a specific analyte from the chromophore perspective, together with its molar-absorption coefficient at the excitation wavelength. In this review we discuss different optical and photothermal methods for measuring Φpl of transparent and scattering systems for the most common classes of luminescent reporters, and critically evaluate their potential and limitations. In addition, reporter-specific effects and sources of uncertainty are addressed. The ultimate objective is to provide users of fluorescence techniques with validated tools for the determination of Φpl, including a series of Φpl standards for the ultraviolet, visible, and near-infrared regions, and to enable better judgment of the reliability of literature data. Graphical Abstractᅟ

New Life of Ancient Pigments: Application in High-Performance Optical Sensing Materials

Calcium, strontium, and barium copper silicates are demonstrated to possess valuable photophysical properties which make them particularly attractive for application in optical chemosensors. Several examples of sensing materials based on these phosphors are provided. Particularly, broad excitation and near-infrared emission makes them ideal candidates for the preparation of ratiometric sensors based on absorption-based indicators. Due to their excellent chemical and photochemical stability and high brightness, these phosphors can serve as reference for fluorescent indicators to enable ratiometric intensity or dually lifetime referenced measurements. Finally, the moderate temperature dependence of the luminescence decay time enables intrinsic temperature compensation of the sensing materials at ambient temperatures. The improved sensitivity at temperatures above 100 °C makes these new materials promising candidates for high-temperature thermographic phosphors.