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Eingeladener Vortrag
- nein (3)
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.
The therapeutic value of antibiotics depends on the susceptibility of the infecting microorganism and the pharmacological profile of the drugs. To assess the value of an antibiotic combination of polymyxin B and miconazole this study examined the in vitro synergistic potential of the two drugs on Gram-negative and Gram-positive bacteria and yeast. Antifungal and antibacterial activity was tested by minimum inhibitory concentration (MIC) of broth macrodilution and urea broth microdilution, by fluorescence microscopy and flow cytometry. Synergism was calculated using the fractional inhibitory concentration index (FICi). With Staphylococcus intermedius as target we found up to an eightfold reduction of the individual MICs when both drugs were combined. However, the FICi was 0.63 suggesting no real interaction between the two drugs. With Escherichia coli, Pseudomonas aeruginosa, and Malassezia pachydermatis as targets the antimicrobial drug combination reduced the MICs of polymyxin B and miconazole from fourfold to hundredfold resulting in FICi between 0.06 and 0.5 which defines a synergistic action. Thus, if polymyxin B and miconazole are combined their effect is greater than the sum of the effects observed with polymyxin B and miconazole independently, revealing bactericidal and fungicidal synergism. Our results indicate a strong therapeutic value for the combination of these antimicrobial agents against Gram-negative bacteria and yeast and a weaker value against Gram positive bacteria for clinical situations where these pathogens are involved.
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.
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.
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.
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.
Aroyl-S,N-ketene acetal-based bichromophores can be readily synthesized in a consecutive three-component synthesis in good to excellent yields by condensation of aroyl chlorides and an N-(p-bromobenzyl) 2-methyl benzothiazolium salt followed by a Suzuki coupling, yielding a library of 31 bichromophoric fluorophores with substitution patterntunable emission properties. Varying both chromophores enables different communication pathways between the chromophores, exploiting aggregation-induced emission (AIE) and energy transfer (ET) properties, and thus, furnishing aggregation-based fluorescence switches. Possible applications range from fluorometric analysis of alcoholic beverages to pH sensors.
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.
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.
We present here the design and characterization of a set of spectral calibration beads. These calibration beads are intended for the determination and regular control of the spectral characteristics of fluorescence microscopes and other fluorescence measuring devices for the readout of bead-based assays. This set consists of micrometer-sized polymer beads loaded with dyes from the liquid Calibration Kit Spectral Fluorescence Standards developed and certified by BAM for the wavelength-dependent Determination of the spectral responsivity of fluorescencemeasuring devices like spectrofluorometers. To cover the wavelength Region from 400 to 800 nm, two new near-infrared emissive dyes were included, which were spectroscopically characterized in solution and encapsulated in the beads. The resulting set of beads presents the first step towards a new platform of spectral calibration beads for the determination of the spectral characteristics of fluorescence instruments like fluorescence microscopes, FCM setups, and microtiter plate readers, thereby meeting the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas, e.g., medical diagnostics. This will eventually provide the basis for standardized calibration procedures for imaging systems as an alternative to microchannel slides containing dye solutions previously reported by us.
Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a 'green' dye and a 'red' dye in a polymer matrix. These dyes present 'Cy3' and 'Cy5' analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.
Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[15N5]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na15NO3-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 µg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 µg/kg fresh weight and 23.9 µg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.
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.
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.
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.
Polypropylene supports were functionalized by plasma-deposition of polymeric allylamine layers. The surface amino groups generated were wet-chemically reacted with xanthene dyes resulting in fluorescent polymer films. The effect of polymer-attachment of the dyes on their emission features was studied fluorometrically and different methods were tested to improve the fluorescence properties of the films. Modification with cucurbit[6]uril (CB6) yields a moderately enhanced fluorescence as well as an improved photostability. The observed effect is most likely due to CB6-induced rigidization of the linker molecules which seems to reduce fluorescence quenching dyedye and fluorophoresurface interactions.
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.
The ever increasing applications of fluorescence techniques in conjunction with the interest in enhanced detection sensitivities in bioanalysis, biosensing, and bioimaging are closely linked to the rational design of novel nontoxic fluorescent nanomaterials with improved brightness and stability that can be reproducibly synthesized from inexpensive starting materials in simple one-pot reactions and easily surface functionalized. This encouraged us to investigate the potential of the commercially available water-dispersible nanoclay Laponite RD with the empirical formula Na0.7(H2O)n{(Li0.3Mg5.5)[Si8O20(OH)4]}, forming 25 nm sized disk-shaped particles, as nanocarriers for different fluorophores. The Si–OH functions at the rims of these disks can be selectively grafted with 3-aminopropyldimethylethoxysilane (APES), thereby enabling subsequent coupling to amine-reactive molecules ranging from target-specific organic ligands and biomolecules to amine-reactive fluorescent labels. Here, we present different strategies for the surface functionalization of nanoclays and the subsequent quantification of the density of synthetically introduced surface amino groups exploiting analytical methods which rely on different detection schemes including elemental analysis, colorimetric assays, and fluorophore labeling strategies. In this respect, we systematically assess the potential of negatively and positively charged, neutral, and zwitterionic dyes to act as fluorescent labels for amino functionalities at the surface of negatively charged nanoclays. Our studies underline the strong influence of dye charge and aggregation tendency on the brightness of the bound dyes and on surface group quantification. Best results regarding surface group analysis and coupling yield were obtained for a neutral dansyl derivative and fluorescamine.
The development of a lanthanum-phosphate glass doped with several rare-earth-ions for use as solid fluorescence standard is described. The cuvette-shaped reference material which shows a characteristic emission intensity pattern upon excitation at 365 nm consisting of a multitude of relatively narrow emission bands in the wavelength region between 450 nm and 700 nm is intended for the day-to-day performance validation of fluorescence measuring devices. Evaluation of the fluorescent glass include the determination of all properties which can affect its relative emission intensity profile or contribute to the uncertainty of the certified values like absorption spectra, fluorescence anisotropy, excitation wavelength and temperature dependence of the spectroscopic features, homogeneity of fluorophore distribution, photo- and long-term stability. Moreover, a certification procedure was developed including the normalization of the intensity profile consisting of several narrow emission bands and the calculation of wavelength-dependent uncertainties. Criteria for the design, characterization, and working principle of the new reference material BAM-F012 are presented, and possible applications of this ready-to-use fluorescence standard are discussed.
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.
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.
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).
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.
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.
Time-resolved flow cytometry
(2019)
The fast identification of a large number of analytes or events is increasingly required in bioanalytical, diagnostic, and security applications. The versatility and straightforward use make multiparametric fluorescence techniques particularly interesting as detection techniques. An established method for high-throughput single-cell and single-particle measurements is flow cytometry (FCM). Using only spectral encoding without further intensity information, state-of-the-art instruments equipped with several light sources and detectors can resolve almost 20 different color codes. However, this is not sufficient to answer complex research questions, e.g. in cell biology and immunology. In contrast, routine applications demand low-cost and sometimes even portable instruments and thus a minimum number of instrument components. Thus, there are currently two main research directions in FCM: the development of methods that can either address increasingly complex analytical challenges or provide low-cost and robust approaches for routine multiplex analyses. Common spectral multiplexing approaches face limitations in both directions. On the one hand, spectral overlap of labels restricts the number of codes and makes elaborate correction schemes necessary. On the other hand, even for lower degrees of multiplexing often a sophisticated optical setup is needed. An alternative to spectral multiplexing and intensity encoding is to exploit the luminescence lifetime (LT) as an encoding parameter. This can allow for extending the parameter space in combination with spectral encoding or result in more simple and compact devices due to fewer optical components. The availability of fast electronics enables miniaturized and portable lifetime measurement setups at relatively low cost. LT-FCM requires to master LT determination with a limited number of detected photons due to the short interaction time of the encoded objects with the laser spot. In this study, we address this issue for time-domain cytometry and present a novel lifetime flow cytometry (LT-FCM) platform based on a compact setup and straightforward time-domain measurements utilizing LT-encoded luminescent beads. Moreover, we present the realization of a first bioanalytical assay with LT-encoded beads.
A new class of boron-dipyrromethene (BDP or BODIPY) dyes was obtained by phenanthrene fusion to the β-pyrrole positions, absorbing in the wavelength range of important laser sources. Despite a 'propeller-like' distorted structure in the crystalline state, the chromophore absorbs (log ε ≥ 5) and fluoresces (Φf ≥ 0.8) strongly and can be easily turned into a fluorescence light-up probe. Incorporation into latex beads produces bright and photostable single-dye and Förster Resonance Energy Transfer (FRET) particles for microscopy applications.
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.
A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.
The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten−horseradish peroxidase conjugate (hapten−HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP−Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG−Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.
Objective: The utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.
Methods: Tumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 mm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with lexc = 488 nm.
Results: Optical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of lmax abs = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at lmax em = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.
Conclusion: Tumor uptake of NaFl leads to changes in the optical properties – a bathochromic shift and broadening of the emission band – possibly caused by the dye’s high pH sensitivity and concentration-dependent reabsorption acting as an innerfilter of the dye’s emission, particularly in the short wavelength region of the Emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.
Merocyanine–triarylamine bichromophores are readily synthesized by sequentially Pd-catalyzed insertion alkynylation–Michael–Suzuki four-component reactions. White-light emissive systems form upon aggregation in 1 : 99 and 0.1 : 99.9 vol% CH2Cl2–cyclohexane mixtures, ascribed to aggregation-induced dual emission (AIDE) in combination with partial energy transfer between both chromophore units as supported by spectroscopic studies.
Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads
(2020)
Semiconductor quantum dots (QDs) embedded into polymer microbeads are known to be very attractive emitters for spectral multiplexing and colour encoding. Their luminescence lifetimes or decay kinetics have been, however, rarely exploited as encoding parameter, although they cover time ranges which are not easily accessible with other luminophores. We demonstrate here the potential of QDs made from II/VI semiconductors with luminescence lifetimes of several 10 ns to expand the lifetime range of organic encoding luminophores in multiplexing applications using time-resolved flow cytometry (LT-FCM). For this purpose, two different types of QD-loaded beads were prepared and characterized by photoluminescence measurements on the ensemble level and by single-particle confocal laser scanning microscopy. Subsequently, these lifetime-encoded microbeads were combined with dye-encoded microparticles in systematic studies to demonstrate the potential of these QDs to increase the number of lifetime codes for lifetime multiplexing and combined multiplexing in the time and colour domain (tempo-spectral multiplexing). These studies were done with a recently developed novel luminescence lifetime flow cytometer (LT-FCM setup) operating in the time-domain, that presents an alternative to reports on phase-sensitive lifetime detection in flow cytometry.
Comparing the photoluminescence (PL) properties of ensembles of nanocrystals like semiconductor quantum dots (QDs) with single particle studies is of increasing interest for many applications of These materials as reporters in bioimaging studies performed under very dilute conditions or even at the single particle level. Particularly relevant is here the PL quantum yield (ΦF), which determines the signal size together with the reporter’s molar extinction coefficient and is a direct measure for nanocrystal quality, especially for the inorganic surface passivation shell and its tightness, which can be correlated also with nanocrystal stability and the possible release of heavy metal ions. Exemplarily for red and green emitting CdTe nanocrystals, we present a method for the determination of ΦF of nanoparticle dispersions at ultralow concentration compared to cuvette measurements using fluorescence correlation spectroscopy (FCS), a single molecule method, and compared to molecular dyes with closely matching spectral properties and known ΦF. Our results underline the potential of this approach, provided that material-inherent limitations like ligand- and QD-specific aggregation affecting particle diffusion and QD drawbacks such as their complex and power-dependent blinking behavior are properly considered as shown here.
To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications.
N-Benzyl aroyl-S,N-ketene acetals can be readily synthesized by condensation of aroyl chlorides and N-Benzyl 2-methyl benzothiazolium salts in good to excellent yields, yielding a library of 35 chromophores with bright solid-state emission and aggregation-induced emission characteristics.
Varying the substituent from electron-donating to electronwithdrawing enables the tuning of the solid-state emission Color from deep blue to red.
We present a comparative study of the spectroscopic properties of the donor–acceptor–donor substituted dyes triphenylamine-allylidenemalononitrile-julolidine (TMJ) and triphenylamine-allylidenemalononitriletriphenylamine (TMT), bearing one and two propeller-like triphenylamine donor moieties, in solvents of varying polarity and viscosity and in the aggregated and solid state. Our results reveal control of the aggregation-induced spectroscopic changes and the packing motifs of the dye molecules in the solid state by the chemical nature and structure of the second nitrogen-containing donor, i.e., a planar and a rigid julolidine or a twisted triphenyl group. Assuming that the TMT and TMJ aggregates show a comparable arrangement of the molecules to the respective crystals, these different molecular interactions in the solid state are responsible for aggregation induced emission (AIE) in the case of TMT and its absence for TMJ. Moreover, a versatile strategy for the fluorescence enhancement of only weakly emissive AIE dyes is shown, turning these dyes into bright nanoscale fluorescent reporters by using them as stains for preformed polymer particles.
Redox switches are applied in various fields of research, including molecular lifts, electronic devices and sensors. Switching the absorbance between UV and Vis/NIR by redox processes is of interest for applications in light harvesting or biomedicine.
Here, we present a series of push-pull benzothiadiazole derivatives with high fluorescence quantum yields in solution and in the crystalline solid state. Spectroelectrochemical analysis reveals the switching of UV-absorption in the neutral state to Vis/NIR absorption in the reduced state. We identify the partial irreversibility of the switching process, which appears to be reversible on the cyclic voltammetry timescale.
The absorption and emission properties of organic dyes are generally tuned by altering the substitution pattern. However, tuning the fluorescence lifetimes over a range of several 10 ns while barely affecting the spectral features and maintaining a moderate fluorescence quantum yield is challenging. Such properties are required for lifetime multiplexing and barcoding applications. Here, we show how this can be achieved for the class of fluoranthene dyes, which have substitution-dependent lifetimes between 6 and 33 ns for single wavelength excitation and emission. We explore the substitution-dependent emissive properties in the crystalline solid state that would prevent applications.
Furthermore, by analyzing dye mixtures and embedding the dyes in carboxyfunctionalized 8 μm-sized polystyrene particles, the unprecedented potential of these dyes as labels and encoding fluorophores for time-resolved fluorescence detection techniques is demonstrated.
Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-Domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.
In this work, we describe a simple solvothermal route for the synthesis of Eu3+-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4−PAA) functionalized with poly(acrylic)acid (PAA), that are applicable as cell labeling probes for multimodal cellular imaging. The Eu3+ doping of the vanadate matrix provides optical functionality, due to red photoluminescence after illumination with UV light. The Gd3+ ions of the nanocrystals reduce the T1 relaxation time of surrounding water protons, allowing these nanocrystals to act as a positive MRI contrast agent with a r1 relaxivity of 1.97 mM−1 s−1. Low background levels of Eu3+, Gd3+, and V5+ in biological systems make them an excellent label for elemental microscopy by Laser Ablation (LA)-ICP-MS. Synthesis resulted in polycrystalline nanocrystals with a hydrodynamic diameter of 55 nm and a crystal size of 36.7 nm, which were further characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and transmission electron microscopy (TEM). The multifunctional nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells (MSCs) and A549 (adenocarcinomic human alveolar basal epithelial) cells.
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.
The fluorescence lifetime is a key property of fluorophores that can be utilized for microenvironment probing, analyte sensing, and multiplexing as well as barcoding applications. For the rational design of lifetime probes and barcodes, theoretical methods have been developed to enable the ab initio prediction of this parameter, which depends strongly on interactions with solvent molecules and other chemical species in the emitters' immediate environment. In this work, we investigate how a conductor-like screening model (COSMO) can account for variations in fluorescence lifetimes that are caused by such fluorophore−solvent interactions. Therefore, we calculate vibrationally broadened fluorescence spectra using the nuclear ensemble method to obtain distorted molecular geometries to sample the electronic transitions with time-dependent density functional theory (TDDFT). The influence of the solvent on fluorescence lifetimes is accounted for with COSMO. For example, for 4-hydroxythiazole fluorophore containing different heteroatoms and acidic and basic moieties in aprotic and protic solvents of varying polarity, this approach was compared to experimentally determined lifetimes in the same solvents. Our results demonstrate a good correlation between theoretically predicted and experimentally measured fluorescence lifetimes except for the polar solvents Ethanol and acetonitrile that can specifically interact with the heteroatoms and the carboxylic acid of the thiazole derivative.