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The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, 13C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network.
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.
With the Calibration Kit Spectral Fluorescence Standards BAM-F001BAM-F005, we developed a simple
tool for the characterization of the relative spectral responsivity and the long-term stability of the emission channel of
fluorescence instruments under routine measurement conditions thereby providing the basis for an improved comparability
of fluorescence measurements and eventually standardization. This first set of traceable fluorescence standards,
which links fluorescence measurements to the spectral radiance scale in the spectral range of 300770 nm and has been optimized for spectrofluorometers, can be employed for different measurement geometries and can be adapted to different fluorescence techniques with proper consideration of the underlying measurement principles.
This report summarizes problems associated with the comparability of measurements of photoluminescence and procedures for the characterization of relevant instruments, focusing on physical and chemical fluorescence standards. To provide recommendations on selecting and using such standards, we derive general and scope-specific requirements and quality criteria for suitable devices and materials and briefly address metrological requirements linked to the realization of comparable measurements. Special emphasis is dedicated to liquid and solid chromophore-based fluorescence standards developed or currently tested by us.
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.
Prerequisite for the rational design of functional organic materials with tailor-made electronic properties is the knowledge of the structure–property relationship for the specific class of molecules under consideration. This encouraged us to systematically study the influence of the molecular structure and substitution pattern of aromatically substituted 1,3,4-oxadiazoles on the electronic properties and packing motifs of these molecules and on the interplay of these factors. For this purpose, seven diphenyl-oxadiazoles equipped with methyl substituents in the ortho- and meta-position(s) were synthesized and characterized. Absorption and fluorescence spectra in solution served here as tools to monitor substitution-induced changes in the electronic properties of the individual molecules whereas X-ray and optical measurements in the solid state provided information on the interplay of electronic and packing effects. In solution, the spectral position of the absorption maximum, the size of Stokes shift, and the fluorescence quantum yield are considerably affected by ortho-substitution in three or four ortho-positions. This results in blue shifted absorption bands, increased Stokes shifts, and reduced fluorescence quantum yields whereas the spectral position and vibrational structure of the emission bands remain more or less unaffected. In the crystalline state, however, the spectral position and shape of the emission bands display a strong dependence on the molecular structure and/or packing motifs that seem to control the amount of dye–dye-interactions. These observations reveal the limited value of commonly reported absorption and fluorescence measurements in solution for a straightforward comparison of spectroscopic results with single X-ray crystallography. This underlines the importance of solid state spectroscopic studies for a better understanding of the interplay of electronic effects and molecular order.
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.
A novel fluorescent host-guest material, molecular sieves of AlPO4-5-type doped with 2,2'-bipyridyl-3,3'-diol, was prepared and spectroscopically characterized. The composite crystals show a pronounced optical anisotropy, indicating a high degree of alignment of the guest molecules within the zeolitic pore system. A mean tilting angle of 22 was found for the orientation of the individual dye molecules in the straight channels. The corrected fluorescence emission spectra were determined, and time-resolved fluorescence studies revealed that the dye molecules are preferentially found in three different types of microenvironment. By invoking pH-dependent studies of the dye in aqueous solution, we could trace these spectroscopic features back to two main influences, coadsorbed water within AlPO4-5 pores and guest-host interactions with a few relatively weak Brnsted acid (defect) sites of the inorganic host network.
The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.
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.
Molecular engineering of polymer surfaces that includes the attachment of functional molecules to existing or previously generated reactive chemical groups like e.g., - OH, - NH2, or - CHO requires simple strategies and tools for the controlled generation of surface functionalities and their derivatization as well as for their identification and eventually quantification. Here, we systematically investigate the plasma-chemical surface modification of polypropylene films in combination with dansyl labeling chemistry and conventional, yet costly, XPS and highly sensitive fluorescence spectroscopy for the detection of surface groups. Based on these results, the potential of and requirements on the fluorometric characterization and quantification of surfaces functionalities are discussed.
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.
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.
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.
A straightforward strategy toward the sensitive fluorometric detection of primary amino groups on plasma-chemically modified polypropylene supports is presented, exploiting the transformation of the sterically nonhindered pyrylium dye Py-1 into its pyridinium counterpart. The reaction-induced blue-shifted absorption and emission bands and an increased fluorescence quantum yield provide the basis for the spectroscopic distinction between covalently bound and free, that is, nonspecifically adsorbed label molecules. With this label, for the first time, plasma-chemically introduced amino functionalities could be monitored on the surface of a polymer film employing fluorescence spectroscopy and confocal laser scanning microscopy.
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.
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.
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 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.
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.
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.
Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all Parameters that can affect the fluorescence properties of the dye-stained beads.
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.
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.
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.
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.
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 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.