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- 2012 (10) (entfernen)
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- Absolute fluorescence quantum yield (1)
- Alexa dyes (1)
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Eingeladener Vortrag
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Targeted ratiometric Nanosensors for the in Vivo Imaging of Tumor Hypoxia in the NIR spectral region
(2012)
Photoluminescence techniques are amongst the most widely used tools in the material and life sciences, with new and exciting applications continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for multiplexing, remote sensing, and miniaturization. Increasing applications of fluorescence techniques in the life sciences and emerging medical applications of fluorescence microscopic techniques including 1P and 2P fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) in e.g. in vivo eye diagnostics boosted the demand for robust, easy-to-use, and reliable fluorescence standards to ensure the reliability and comparability of fluorescence data. This includes fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and instrument performance validation, fluorescence intensity standards for the quantification of measured intensities and for signal referencing, and lately, also fluorescence lifetime standards.
Synthesis and characterisation of highly fluorescent core-shell nanoparticles based on Alexa dyes
(2012)
Current and future developments in the
emerging field of nanobiotechnology are closely
linked to the rational design of novel fluorescent
nanomaterials, e.g. for biosensing and imaging applications.
Here, the synthesis of bright near infrared
(NIR)-emissive nanoparticles based on the grafting
of silica nanoparticles (SNPs) with 3-aminopropyl
triethoxysilane (APTES) followed by covalent attachment
of Alexa dyes and their subsequent shielding by an
additional silica shell are presented. These nanoparticles
were investigated by dynamic light scattering (DLS),
transmission electron microscopy (TEM) and fluorescence
spectroscopy. TEM studies revealed the monodispersity
of the initially prepared and fluorophorelabelled
silica particles and the subsequent formation of
raspberry-like structures after addition of a silica
precursor.Measurements of absolute fluorescence quantum
yields of these scattering particle suspensions with
an integrating sphere setup demonstrated the influence
of dye labelling density-dependent fluorophore aggregation
on the signaling behaviour of such nanoparticles.
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 (19F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF3 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.
In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles.
One of the most active research areas in the life and material sciences is the design and synthesis of fluorescent nano- and micrometre sized particles for applications e.g. as labels, sensor systems, and platforms for fluorescence assays or barcoding materials. The reliable and reproducible fabrication of such particles as well as many applications require accurate, simple, and versatile procedures for the determination of the dye content per particle which affects e.g. the brightness of these materials and their surface charge and thus, colloidal stability. Here, four fast and inexpensive spectroscopic methods for the quantification of the fluorophore content of beads are presented and compared for nanometre- and micrometre sized polystyrene particles loaded or labeled with commercial fluorophores, differing in dye class, charge, and hydrophilicity. This included the determination of the amount of incorporated dye from absorption spectra of bead suspensions, via dissolving of the polymer matrix, via extraction of the polymer matrix, and from the supernatant of the swelling solution or reaction mixture. Method validation was performed with a sulfur-containing dye and elemental analysis. Based upon this method comparison and the accomplishable uncertainties, two reliable strategies for particle characterization and bead process control are identified that can be easily extended to other materials.