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Organisationseinheit der BAM
To improve the reliability of fluorescence data in the life and material sciences and to enable accreditation of fluorescence techniques, standardization concepts are required that guarantee and improve the comparability of fluorescence measurements. At the core of such concepts are simple and evaluated fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and for instrument performance validation (IPV). Similarly in need are fluorescence intensity standards for the quantification from measured intensities and for signal referencing, thereby accounting for excitation light-induced intensity fluctuations. These standards should be preferably certified, especially for use in regulated areas like medical diagnostics. This encouraged us to develop liquid and solid standards for different fluorescence parameters and techniques for use under routine measurement conditions in different formates. Special emphasis was dedicated to the determination and control of the spectral responsivity of detection systems, wavelength accuracy, homogeneity of illumination, and intensity referencing for e.g. spectrofluorometers, fluorescence sensors and confocal laser scanning fluorescence microscopes. Here, we will present design concepts and examples for mono- and multifunctional fluorescence standards that provide traceability to radiometric units and present a first step towards a toolbox of standards.
Unter dem Motto „Innovation und Qualitätssicherung in der (Bio)Analytik“ werden in der Arbeitsgruppe Fluoreszenzspektroskopie der BAM, Bundesanstalt für Materialforschung und -prüfung, funktionelle Chromophor-Systeme, einfache Signalverstärkungs- und Multiplexingstrategien sowie innovative Validierungs- und rückführbare Standardisierungskonzepte für verschiedene fluorometrische Messgrößen und Methoden entwickelt. Im Mittelpunkt stehen dabei molekulare Fluorophore, Nanokristalle mit größenabhängigen optischen Eigenschaften (sogenannte Quantenpunkte, QDs) und fluoreszierende Partikel variabler Größe sowie Sonden und Sensormoleküle für neutrale und ionische Analyte und für die Charakterisierung von funktionellen Gruppen. Dabei erfolgen auch methodische Entwicklungen für die Fluoreszenzspektroskopie, die Fluoreszenzmikroskopie, die Milcrofluorometrie, die Sensorik und die Mikroarraytechnologie. Ziele sind u. a. das Design und die Untersuchung von multiplexfähigen selektiven und sensitiven Sonden für die Biomarkeranalytik, die Entwicklung von Methoden zur Charakterisierung der signalrelevanten Eigenschaften dieser Chromophor-Systeme und zur Charakterisierung von funktionellen Gruppen an Oberflächen und ihre Validierung sowie die Entwicklung und Bereitstellung von formatadaptierbaren, flexibel ersetzbaren
Standards für die fluoreszenzbasierte Multiparameteranalytik.
The widespread use and acceptance of fluorescence techniques especially in regulated areas like medical diagnostics is closely linked to standardization concepts that guarantee and improve the comparability and reliability of fluorescence measurements. At the core of such concepts are dependable fluorescence standards that are preferably certified. The ever rising interest in fluorescence measurements in the near-infrared (NIR) spectral region renders the availability of spectral and intensity standards for this wavelength region increasingly important. This encouraged us to develop approaches to solid NIR standards based upon dye-doped polymers and assess their applicationrelevant properties in comparison to metal ion-doped glasses. The overall goal is here to provide inexpensive, easily fabricated, and robust internal and external calibration tools for a broad variety of fluorescence instruments ranging e.g. from spectrofluorometers over fluorescence microscopes to miniaturized fluorescence sensors.
Increasing the information content from bioassays which requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement is an important field of research, especially in the context of meeting current security and health concerns. An attractive alternative to spectral multiplexing, which relies on fluorescent labels excitable at the same wavelength, yet sufficiently differing in their emission spectra or color presents lifetime multiplexing. For this purpose, we recently introduced a new strategy based on 'pattern-matching' in the lifetime domain, which was exemplary exploited for the discrimination between organic dyes and quantum dot labels revealing multi-exponential decay kinetics and allowed quantification of these labels. Meanwhile, we have succeeded in extending this lifetime multiplexing approach to nanometer-sized particle labels and probes absorbing and emitting in the visible (vis) and near-infrared (NIR) spectral region. Here, we present a first proof-of-principle of this approach for a pair of NIR-fluorescent particles. Each particle is loaded with a single organic dye chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics. Examples for the lifetime-based distinction between pairs of these fluorescent nanoparticles in solution and in cells are presented. The results underline the potential of fluorescenc lifetime multiplexing in life science and bioanalysis.
A series of twelve 3-piperazinyl propenylidene indolone merocyanines was synthesized in a one-pot fashion using a consecutive three-component insertion-coupling-Michael addition sequence. Physicalorganic treatment of the absorption data of a consanguineous series of this library allows semiquantitative Linear Free Energy Relationships (LFERs) to be established and confirmation of the positive Absorption solvatochromicity. All Boc-substituted piperazinyl merocyanines display aggregation induced Emission (AIE), which was corroborated for two solvent systems. In particular, crystallization-induced Emission enhancement (CIEE) induced by ultrasonication could be shown for a model chromophore by confocal laser scanning microscopy (CLSM).
Photoluminescence techniques are amongst the most widely used Tools in the life sciences, with new and exciting applications in medical diagnostics and molecular Imaging continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for Multiplexing, remote sensing, and miniaturization. General drawbacks are, however, signals, that contain unwanted wavelength- and polarization contributions from Instrument-dependent effects, which are also time-dependent due to aging of Instrument-components, and difficulties to measure absolute flourescence entensities. Moreover, scattering Systems require Special measurement geometries and the interest in new optical Reporters with Emission > 1000 nm strategies for reliable measurements in the second diagnostic for the comparison of material Performance and the rational designg of new flourophores with improved properties.
Here, we present strategies to versatile method-adaptable liquid and solid flourescence Standards for different flourescence paramters including traceable Instrument calibration procedures and the design of integrating spere setups for the absolute measurements of emission spectra and Quantum yields in the wavelength Region of 350 to 1600 nm. Examples are multi-Emitter glasses, spectral flourescence Standards, and quantum yield Standards for the UV/vis/NIR.
Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding.
Aggregation-induced emission (AIE) has been meanwhile observed for many dye classes and particularly for fluorophores containing propeller-like groups. Herein, we report on the AIE characteristics of a series of four hydrophobic pyrrolidinylvinylquinoxaline (PVQ) derivatives with phenyl, pyrrolyl, indolyl, and methoxythienyl substituents used to systematically vary the torsion angle between this substituent at the quinoxaline C2 position and the planar PVQ moiety. These molecules, which are accessible via four- or five-component one-pot syntheses, were spectroscopically studied in organic solvents and solvent−water mixtures, as dye aggregates, solids, and entrapped in polystyrene particles (PSP). Steady-state and time-resolved fluorescence measurements revealed a strong fluorescence enhancement for all dyes in ethanol−water mixtures of high water content, accompanying the formation of dye aggregates with sizes of a few hundred nm, overcoming polarity and H-bonding-induced fluorescence quenching of the chargetransfer-type emission of these PVQ dyes. The size and shape of these dye aggregates and the size of the AIE effect are controlled by the water content and the substituent-dependent torsion angle that influences the nucleation process and the packing of the molecules during aggregation. Staining of 1 μm-sized carboxy-functionalized PSP with the PVQ dyes resulted also in a considerable increase in the fluorescence quantum yield and lifetime, reflecting the combined influence of the restricted molecular motion and the reduced polarity of the dye microenvironment.
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.
The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.
Water-dispersible and (bio)functionalizable nanoclays have a considerable potential as inexpensive carriers for organic molecules like drugs and fluorophores. Aiming at simple design strategies for red-emissive optical probes for the life sciences from commercial precursors with minimum synthetic effort, we systematically studied the dye loading behavior and stability of differently functionalized laponites. Here, we present a comprehensive study of the absorption and emission properties of the red emissive hydrophobic and neutral dye Nile Red, a well-known polarity probe, which is almost insoluble and nonemissive in water. Adsorption of this probe onto disk-shaped nanoclays was studied in aqueous dispersion as function of dye concentration, in the absence and presence of the cationic surfactant cetyltrimethylammonium bromide (CTAB) assisting dye loading, and as a function of pH. This laponite loading strategy yields strongly fluorescent nanoclay suspensions with a fluorescence quantum yield of 0.34 at low dye loading concentration. The dye concentration-, CTAB-, and pH-dependent absorption, fluorescence emission, and fluorescence excitation spectra of the Nile-Red6#8211;nanoclay suspensions suggest the formation of several Nile Red species including emissive Nile Red monomers facing a polar environment, nonemissive H-type dimers, and protonated Nile Red molecules that are also nonfluorescent. Formation of all nonemissive Nile Red species could be suppressed by modification of the laponite with CTAB. This underlines the great potential of properly modified and functionalized laponite nanodisks as platform for optical probes with drug delivery capacities, for example, for tumor and therapy imaging. Moreover, comparison of the Nile Red dimer absorption spectra with absorption spectra of previously studied Nile Red aggregates in dendrimer systems and micelles and other literature systems reveals a considerable dependence of the dimer absorption band on microenvironment polarity which has not yet been reported so far for H-type dye aggregates.
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.
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.
Encapsulation of hydrophobic dyes in polystyrene micro- and nanoparticles via swelling procedures
(2011)
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, aminomodified
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.