Spectroscopic characterization of coumarin-stained beads: quantification of the number of fluorophores per particle with solid-state 19F-NMR and measurement of absolute fluorescence quantum yields.
ABSTRACT The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative ((19)F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF(3) group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.
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ABSTRACT: The surface of poly(methyl methacrylate) particles with different amounts of a grafted layer of poly(acrylic acid) was labeled with varying degrees of an amino derivative of fluorescein isothiocyanate. The resulting fluorescent polymer particles were analyzed by absorption spectroscopy and by steady-state and time-resolved fluorescence spectroscopy including measurements of the fluorescence anisotropy. The combined results indicate that the overall decrease in fluorescence intensity with increasing surface concentrations of the fluorophore can be traced back to the formation of non-fluorescent aggregates. A mechanism is proposed, in which the excitation energy migrates between identical fluorophores until it is transferred to non-fluorescent aggregates acting as an energy trap. Increases in the surface fluorophore concentration increase both the probability for energy transfer between identical fluorophores and the probability for energy transfer to non-fluorescent aggregates. Furthermore, we suggest that this mechanism also applies to fluorescent protein conjugates and rationalizes the nonlinear dependence of the fluorescence emission on the labeling density.Photochemical and Photobiological Sciences 01/2013; · 2.92 Impact Factor
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ABSTRACT: 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.Nature Protocol 08/2013; 8(8):1535-50. · 8.36 Impact Factor
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ABSTRACT: Acrylic acid 3-acetyl-2-oxo-2 H-chromen-7-yl ester (ACA) was rationally designed and synthesized as a simple and effective fluorescent probe for sensing cysteine with high selectivity and naked-eye detection. The probe can detect cysteine by fluorescence spectrometry with a detection limit of 0.657 μM and can be used with calf serum and in live cell imaging. The conjugate addition/cyclization sequence mechanism of the reaction between ACA and cysteine was conﬁrmed by ESI-MS and fluorescence spectra.Biosensors & Bioelectronics 01/2014; 59:35–39. · 6.45 Impact Factor