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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).
Plasmapolymerisierte Allylaminschichten (50 nm) wurden auf Polypropylenfolien abgeschieden. Die auf diese Weise generierte NH2-funktionalisierte Polymeroberfläche wurde mit Xanthen-Fluorophoren chemisch umgesetzt. Da die Farbstoff-gekoppelten Folien nur geringe Fluoreszenz aufwiesen, wurden zwei Wege zur möglichen Entkopplung der Farbstoffe von der Oberfläche und damit zur Erhöhung der Fluoreszenzintensität getestet. Als erstes wurde ein Spacer durch die Umsetzung der primären Aminogruppen mit Glutaraldehyd und der nachfolgenden Kettenverlängerung mit Diaminohexan eingeführt. Diese nachfolgende Reaktion ergab eine spacergebundene NH2-Oberfläche, die analog der Allylaminschicht mit den Farbstoffen umgesetzt wurde. Als weiterer Weg wurde der Zusatz einer geeigneten Käfigverbindung, des Cucurbit[6]urils, getestet. Die spacergebundenen und zusätzlich mit Cucurbituril modifizierten aminofunktionellen Oberflächen zeigten nach der Farbstoffankopplung eine verbesserte Fluoreszenzintensität.
Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools, detection methods and imaging applications for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for steady state fluorescence diagnostics, a method having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers such as absorption/excitation cross sections and quantum yield. This has been done for different types of dopands in different materials such as glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Samples doped with several fluophores of different emission wavelengths and decay times are required for fluorescent multiplexing applications. Decay times shorter than 100 ns are of special interest. In addition, a proper knowledge is necessary of quantum efficiency in highly scattering media. Recently, quantum efficiency in YAG:Ce glass ceramics has been successfully investigated. Glass and glass ceramics doped with threefold charged rare earth elements are available. However, these samples have the disadvantage of emission decay times much longer than 1 microsecond, due to the excitation and emission of their optical forbidden electronic transitions. Therefore first attempts have been made to produce decay-time standards based on organic and inorganic fluophores. Stable LUMOGEN RED pigments and YAG:Ce phosphors are diluted simultaneously in silicone matrices using a wide range of concentrations between 0.0001 and 2 wt%. Organic LUMOGEN RED has decay times in the lower nanosecond range with a slight dependency on concentration and temperature. In addition, the well-known decay properties of inorganic YAG:Ce are observed also embedded in silicone matrix. Luminescent silicone layers are obtained with thicknesses between 150 and 300 m and no change of decay time, which has been determined to be between 60 and 62 ns. Finally, first results are shown for fluorescent CaF2:Pb glass ceramics embedded in a silicate glass matrix. Wavelength accuracy and lifetime are characterized for different environmental conditions such as temperature treatment and UV irradiation. Moreover, intensity patterns, e.g. line profiles and results, are discussed on homogeneity and photo and thermal stability, respectively. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy are employed as diagnostic methods to get a microscopic view of the relevant physical processes. The work is funded by BMBF under project number 13N8849.
In all fluorescence-based techniques, the measured signals contain not only sample-related but also instrument-specific contributions, which limit the direct comparison of fluorescence data obtained e.g. on different devices or at different times and often hamper quantification. To rule out instrumentation as major source of variability of emission data, accepted fluorescence standards and procedures for the control of instrument specifications and long-term performance are required. For flow cytometry (FCM), a broad variety of fluorophore-stained polymer beads differing in emission wavelength and intensity is available for the testing of the alignment, sensitivity, and other parameters of FCM. These calibration tools are intended to facilitate the assessment of instrument performance to ensure reliable measurements and to improve the comparability of FCM experiments.
As a step towards an improved comparability of fluorescence data, with special emphasis on spectroscopic methods measuring nano- and micrometer-sized fluorescent objects, we are currently developing a set of fluorescent polystyrene (PS) beads loaded with luminophores from the certified BAM-Kit “Spectral fluorescent standards”, initially developed for the calibration of fluorescence spectrometers. Here, we present first results from studies of these fluorophore-loaded polymer beads. Moreover, new beads are made to supplement this kit by encapsulating near-infrared (NIR)-emissive luminophores in PS beads to cover the UV/VIS, and NIR wavelength range.
These beads are designed for calibration of flow cytometers and other fluorescence imaging systems to meet the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas like e.g. medical diagnostics.
The aim of this article is to illustrate the need for an improved quality assurance in fluorescence microscopy. From the instrument-side, this can be achieved by a better understanding, consideration, and regular control of the instrument-specific parameters and quantities affecting measured fluorescence signals. Particularly, the need for requirements on physical- and chemical-type instrument standards for the characterization and performance validation of spectral fluorescence microscopes (SFMs) is discussed and suitable systems are presented. Special emphasis is given to spectral fluorescence standards and to day-to-day intensity standards for SFMs. Fluorescence standards and well-characterized fluorescence microscopes are the first and essential steps towards the comparability and the understanding of the variability in fluorescence microscopy data in medical and life sciences. In addition, standards enable the distinction between instrument-specific variations and fluorescent label- or probe-related uncertainties as well as generally sample-related effects.
There is an increasing interest in bridging the gap between the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) commonly assessed in ensemble studies and the PL features of single QDs for life sciences applications such as bioimaging studies or use in microfluidic assays. The fluorescence quantum yield (ΦF) is a key performance parameter for all molecular and nanoscale emitters, increasingly employed in nanoscience, nanotechnology, and medical diagnostics.
ΦF determines not only the signal size together with the reporter´s molar extinction coefficient, yet it is particularly relevant for nanocrystals like QDs with coordinatively bound surface ligands and size- and surface chemistry-dependent PL characteristics.
The importance of ΦF measurements at ultralow concentration encouraged us to explore the potential of fluorescence correlation spectroscopy (FCS) for the relative determination of ΦF of ligand-stabilized CdTe nanocrystals in comparison to molecular dyes with closely matching spectral properties and known ΦF.
We describe a FCS-based method for the relative determination of ΦF of dispersed QDs at ultralow concentrations, and procedures to overcome QD-inherent challenges like complex and power-dependent blinking behavior as well as ligand- and QD-specific aggregation. We could demonstrate the potential of this approach by comparison with steady state ensemble measurements.
Die Entwicklung von Hybridmaterialien mit stark verbesserten Funktionen gelingt durch die Kombination geeigneter Nanomaterialien als Träger mit Motiven aus der supramolekularen Chemie. Diese hetero-supramolekularen Konzepte bieten vielfältige Möglichkeiten, die Lücken zwischen der Molekülchemie, Materialwissenschaften und Nanotechnologie zu schließen. Im Hinblick auf funktionelle Aspekte wurden in jüngster Zeit wichtige Fortschritte erzielt, z. B. verbesserte Erkennungs- und Sensoreigenschaften durch die Anordnung von Molekülen auf vororganisierten Oberflächen, der reversible Aufbau von nanometergroßen Netzwerken und dreidimensionalen Strukturen oder biomimetische und gesteuerte chemische Prozesse in Hybrid-Nanomaterialien, die als Grundlage für hoch spezialisierte Protokolle in dreidimensionalen Gerüststrukturen dienen können. Diese Ansätze ermöglichen eine Feinabstimmung der Eigenschaften von Nanomaterialien und eröffnen neue Perspektiven für die Anwendung supramolekularer Konzepte.
Luminescence techniques are amongst the most commonly used analytical methods in life and material sciences due to their high sensitivity, nondestructive character, and easy instrumentation suitable for miniaturization. Photoluminescence signals are, however, affected by wavelength-, polarization-, and time-dependent instrument-related effects. Thus, at the core of standardization approaches for all fluorescence-based techniques are evaluated fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and for instrument performance validation (IPV).
Here, we summarize the research of BAM division 1.2 on development of liquid and solid fluorescence standards for various application-relevant fluorescence parameters and techniques. The portfolio of BAM fluorescence reference materials presently consists of:
i) a Spectral Fluorescence Standard Kit, i.e., a set of liquid fluorescence standards with certified normalized corrected emission spectra, for the determination of a broad variety of fluorescence parameters
ii) a ready-to-use, glass-based multi-emitter fluorescence standard for IPV and the determination of instrument-to-instrument variations
iii) specially adapted calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control
iv) quantum yield (Φf) standards for relative determination of the key performance parameter Φf of fluorescent materials, which can be also used for the evaluation of the performance of absolute, standard-free methods utilizing integrating sphere setups or spectrometer accessories. These materials, that will eventually cover the ultraviolet, visible, and near infrared spectral region, are currently under certification.
This toolbox of method-adapted reference materials can perfectly complement existing fluorescence standards. These easy-to-use, reference materials can pave the way to traceable fluorescence measurements to a radiometric scale like the spectral radiance or spectral photon radiance for all users of fluorescence techniques in material sciences and analysis, as well as environmental monitoring and biotechnology. They are particularly useful for customers working in strongly regulated areas like medical diagnostics or pharmaceutical research, where certified standards in conjunction with validated standard operating procedures are mandatory.
Fluorescence microscopic and spectroscopic monitoring of degradation processes upon polymer ageing
(2017)
The majority of all routinely used methods to assess polymer aging are based on destructive tests and methods. Early indicators for the deterioration of polymer materials are e.g., physical or mechanical properties like tensile strength, adhesion, brittleness, and color. It is well-known, however, that predominantly chemical changes are the underlying process of the physical changes that occur in organic materials upon aging over time. Typical initial steps during polymer degradation are crosslinking or chain breaking, alteration of autofluorescence, “yellowing” or bleaching caused by the formation of new functional groups. A straightforward strategy towards the sensitive detection and monitoring of chemical changes in the course of polymer aging is based on non-destructive optical measurements. Luminescence techniques, one of the most sensitive spectroscopic methods are the method of choice. Here, we present first results of luminescence-based monitoring of polymer degradation induced by different environmentally relevant weathering factors (e.g. humidity and UV exposure). Our studies include fluorescence spectroscopy as well as spectral scanning confocal fluorescence microscopy and clearly demonstrate the possibility to follow accelerate-aging processes by luminescence detection.
Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers like absorption/excitation cross sections and quantum yield. This can be done for different types of dopands in different materials like glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Based on the optical spectroscopy data we will discuss options for characteristic doped glasses and glass ceramics with respect to scattering and absorption regime. It has shown recently for YAG:Ce glass ceramics that for a proper determination of the quantum efficiency in these highly scattering media a reference material with similar scattering and fluorescent properties is required. This may be performed using the emission decay measurement diagnostics, where the decay time is below 100 ns. In this paper we present first results of these aspects using well performing LUMOGEN RED organic pigments for a comparison of mainly transparent glass with glass ceramics doped with various amounts of dopands e.g. ions of raw earth elements and transition metals. The LUMOGEN red is embedded in silica and polyurethane matrices. Characterisations on wavelength accuracy and lifetime for different environmental conditions (temperature, UV irradiation) have been performed. Moreover intensity patterns and results for homogeneity, isotropy, photo and thermal stability will be discussed. In a next step we will show the transfer of the characterisation methods to inorganic fluophores (YAG:Ce) in silicon. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy working in different temperature regimes (10 - 350 K) are employed diagnostic methods in order to get a microscopic view of the relevant physical processes and to prove the correctness of the obtained data. The work is funded by BMBF under project number 13N8849.
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.
Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and biotechnical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. For routine measurements by fluorescence techniques the existence of an improved quality assurance is one of the basic needs. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values only. Typical requirements onto fluorescence reference materials or standards deal with the verification of the instrument performance as well as the improvement of the data comparability. Especially for biomedical applications fluorescence labels are used for the detection of proteins. In particular these labels consist of nano crystalline materials like CdS and CdSe. The field of Non-Cadmium containing materials is under investigation. In order to evaluate whether glass based materials can be used as standards it is necessary to calculate absolute values like absorption/excitation cross sections or relative quantum yields. This can be done using different quantities of dopands in glass, glass ceramics or crystals. The investigated materials are based on different types of glass, silicate, phosphate and boron glass, which play a dominant role for the absorption and emission mechanism. Additional to the so-called elementary fluorescence properties induced by raw earth elements the formation of defects lead to higher cross sections additionally. The main investigations deal with wavelength accuracy and lifetime of doped glasses, glass ceramics and crystalline samples. Moreover intensity patterns, homogeneity aspects and photo stability will be discussed.
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.
Problems associated with the measurement of photoluminescence are briefly reviewed, including relevant instrument parameters affecting these measurements. Procedures for the characterization of relevant instruments are discussed, focusing on spectrofluorometers, and fit-for-purpose methods including suitable standards are recommended. The aim here is to increase the awareness of the importance of reliable instrument characterization and to improve the comparability of measurements of photoluminescence.
El desarrollo de receptores sintéticos complejos basados en conceptos de la química supramolecular no garantiza en ocasiones un reconocimiento molecular selectivo. Una alternativa en ciertos aspectos más simple y prometedora es el empleo de materiales híbridos orgánico-inorgánicos preparados mediante la funcionalización de sólidos inorgánicos porosos con receptores sintéticos adecuados. El anclaje de estos sistemas coordinantes a un soporte sólido da lugar a ciertos efectos sinérgicos que no están presentes ni en el material inicial ni en el receptor por separado y que sugieren que estos nuevos materiales híbridos pueden ser empleados en el desarrollo de nuevos sensores y nuevos protocolos de reconocimiento molecular y/o iónico.
The development of synthetic receptors based in supramolecular chemistry concepts will no allow a selective molecular recognition. A more simple, convenient and innovative approach relies on the use of organic-inorganic hybrid materials. These hybrid materials will be prepared by the grafting of certain synthetic receptors onto porous inorganic solids. The grafting of these coordinating systems onto a solid support leads to synergic effects that are hardly achievable with only the synthetic receptors or the solids alone. The presence of these synergic effects suggests that these organic-inorganic hybrid materials will be used for the development of novel sensory systems and novel molecular and/or ionic recognitionprotocols.
The overall interest in nanotoxicity, triggered by the increasing use of nanomaterials in the material and life sciences, and the synthesis of an ever increasing number of new functional nanoparticles calls for standardized test procedures and for efficient approaches to screen the potential genotoxicity of these materials. Aiming at the development of fast and easy to use, automated microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the detection of DNA double strand breaks (DSB) as a sign for genotoxicity. Here, we provide first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability and different cell lines like Hep-2 and 8E11 cells, which reveal a dependence of the genotoxicity on the chemical composition as well as the surface chemistry of these nanomaterials. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.
Luminescence techniques are amongst the most commonly used analytical methods in the life and the material sciences due to their sensitivity and nondestructive character. All photoluminescence signals are, however, affected by wavelength-, polarization- and time-dependent instrument-related effects. Furthermore, substantial challenges to measure absolute luminescence intensities complicate the comparison of data recorded with different instruments and on the same instrument at different times. These problems can be easily resolved with fluorescence standards used for instrument performance validation (IPV) and determination of instrument-to-instrument variations, which allow to measure, quantify, and monitor the wavelength-dependent spectral responsivity for typically used instrument settings. For example, a set of liquid fluorescence standards, the BAM Kit F001-F005, and a ready-to-use glass-based fluorescence standard BAM F-012 developed and certified by BAM enable the characterization of many fluorescence parameters in the UV/vis wavelength range. For the increasingly used near infrared (NIR) region, standards and calibration tools are still very rare. Reliable spectral fluorescence standards and intensity or quantum yield standards are currently not available for the NIR, even though in biology, molecular imaging, and clinical diagnostics fluorescence labels absorbing and emitting in the long wavelength region beyond 650 nm are being increasingly used.
This limitation hampers the reliability and comparability of fluorescence measurements in the NIR and calls for simple fluorescence standards for instrument characterization and for the quantification of fluorescence intensities and efficiencies to improve the comparability of the emission measurements in the NIR. This encouraged us to assess the potential of several NIR-emitting materials as spectral fluorescence standards, thereby extending the BAM Kit from the UV/vis into the NIR up to 950 nm. Moreover, we currently certify quantum yield standards for the UV/vis/NIR to improve the reliability of relative measurements of this spectroscopic key quantity particularly > 650 nm. These tools enable an instrument characterization, signal referencing, quality assurance, traceability, and method validation now also for wavelengths > 650 nm, thereby improving the reliability of fluorescence data in pharmaceutical research, medical and clinical diagnostics, material analysis, and environmental monitoring.
Here, we summerize our efforts concerning new design concepts and examples for fluorescence standards that can provide traceability to radiometric units and present a first step towards a toolbox of fluorescence standards, currently consisting of:
i) A first set of liquid fluorescence standards enables the determination of a broad variety of fluorescence parameters was developed and certified by BAM and is distributed by Sigma-Aldrich.
ii) Ready-to-use, glass-based fluorescence standards for instrument performance validation (IPV) and determination instrument-to-instrument variations can also be used as wavelength standard for fluorescence instruments with low requirements on spectral resolution and allow monitoring of temporal changes of the wavelength-dependent spectral responsivity.
iii) Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.
iv) We currently develop reference materials, which can be used as reliable quantum yield standards for relative methods for the determination of QY and can be valuable in the evaluation of the performance and sources of uncertainty of absolute, standard-free methods using e.g. integrating spheres.
The potential of spectrofluorometry and fluorescence microscopy for the characterization and quantification of different functionalities like OH and NH2 groups at plasma-chemically modified polymer surfaces is assessed using traditional reactive dyes such as dansyl derivatives and a sophisticated VIS-excitable chromogenic and fluorogenic pyrylium label showing binding-induced spectral and intensity changes in absorption and emission. Aiming at an improved fluorometric surface analysis, based upon these measurements, several sources of uncertainty inherent to fluorescence measurements are illustrated ranging from environment-dependent dye absorption and emission features over spectral correction and nonspecific adsorption to the critical influence of label choice on the measured background. Solutions to these drawbacks are given thereby underlining the potential of fluorometry for surface analysis.
Polymer surfaces were modified in low-pressure glow discharge plasmas for introduction of monotype functional groups of different type and density. For this purpose three ways are selected, (i) oxygen plasma treatment followed by wet-chemical reduction of O functional groups to OH groups, (ii) plasma bromination for introducing C - Br groups and (iii) coating by deposition of thin plasma (co-) polymerized layers of functional groups-bearing monomers with OH, NH2, COOH, epoxy etc. functionalities. Subsequently, these groups were used as anchoring points for chemical grafting of spacer molecules, oligomers, prepolymers, fluorescent labels, ionic and nucleic acid residues, employing different chemical routes. The yield in monosort functional groups at polymer surfaces ranged from 1014 (process i), 2040 (process ii) and 1831 groups per 100 C atoms (process iii) as measured by XPS after derivatization. The consumption of functional groups amounted to 4090% of all functionalities present at the surface and depended on the dimensions of grafted molecules. For infinitely variably tuning the number of functional groups process iii was performed as copolymerization of a functional group-carrying comonomer with a non-functionalized (chain-extending) comonomer.