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Between Aromatic and Quinoid Structure: A Symmetrical UV to Vis/NIR Benzothiadiazole Redox Switch
(2020)
Reversibly switching the light absorption of organic molecules by redox processes is of interest for applications in sensors, light harvesting, smart materials, and medical diagnostics. This work presents a symmetrical benzothiadiazole (BTD) derivative with a high fluorescence quantum yield in solution and in the crystalline state and shows by spectroelectrochemical analysis that reversible switching of UV absorption in the neutral state, to broadband Vis/NIR absorption in the 1st oxidized state, to sharp band Vis absorption in the 2nd oxidized state, is possible.
For the one-electron oxidized species, formation of a delocalized radical is confirmed by electron paramagnetic resonance spectroelectrochemistry. Furthermore, our results reveal an increasing quinoidal distortion upon the 1st and 2nd oxidation, which can be used as the leitmotif for the development of BTD based redox switches.
The basics of absorption spectroscopy (termed also photometry) will be presented with focus on transparent solutions of different molecular and nanocrystalline absorbers and the ultraviolet (UV), visible (vis), and near-infrared (NIR) spectral region. Thereby, also typical sources of uncertainty will be addressed. Subsequently, several examples for typical applications of absorption measurements in the life and material sciences will be briefly shown ranging from aggregation studies and dye labeling densities of biomolecules (dye-to-biomolecule ratios) over optical assays for thiol and protein quantification to the optical determination of the size of semiconductor nanocrystals using size curves.
A brief introduction to fluorescence spectroscopy will be provided, ranging from typically measured fluorescence quantities over instrument-specific contributions to measured fluorescence signals to selected applications. In this context, an overview of the photoluminescence properties of molecular and nanoscale luminescence reporters will be given including a brief insight into their photophysics and fluorescence standards designed by division Biophotonics for the calibration and instrument performance validation of fluorescence measuring devices will be presented.
A brief introduction to fluorescence spectroscopy will be provided, ranging from typically measured fluorescence quantities over instrument-specific contributions to measured fluorescence signals to selected applications. In this context, an overview of the photoluminescence properties of molecular and nanoscale luminescence reporters will be given including a brief insight into their photophysics and fluorescence standards designed by division Biophotonics for the calibration and instrument performance validation of fluorescence measuring devices will be presented.
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.
Research of division Biophotonics at the Federal Institute for Materials Research and Testing (BAM) covers several topics including photophysics of molecular and nanocrystalline emitters, the development of signal enhancement, multiplexing, and barcoding strategies, surface group quantification, the rational design of different types of stimuli-responsive optical probes, and concepts and reference materials for the validation of optical-spectroscopic measurements. In the following representative examples for each of these topics are given. Also, current developments like single particle spectroscopy and flow cytometry with lifetime detection and newly certified fluorescence quantum yield standards are presented.
Tricalcium aluminate (C3A) is found with less than 10% wt. of the total composition; however, during hydration, C3A plays an important role in the early hydration of cement in the presence of gypsum as a set retarder.
The aim of this investigation is to assess the suitability of optical spectroscopy and a dye-based optical probe to monitor early hydration of C3A in the presence of gypsum and hemihydrate. Optical evaluation was performed using steady-state fluorescence and diffuses reflectance spectroscopy (UV-VisDR). Phase characterization during hydration was done with in-situ X-ray diffraction. UV-VisDR with a cyanine dye probe was used to monitor the formation of metastable phases and was employed together with fluorescence spectroscopy, to follow the Aggregation and disaggregation of the dye during hydration. In conclusion, for the first time, a cyanine dye was identified as a feasible and stable probe to monitor C3A hydration changes in the presence of calcium sulfate.
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.
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 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.
We report on the temperature- and structural-dependent optical properties and photophysics of a set of boron dipyrromethene (BODIPY) dyes with different substitution patterns of their meso-aryl subunit. Single-crystal Xray diffraction analysis of the compounds enabled a classification of the dyes into a sterically hindered and a unhindered group. The steric hindrance refers to a blocked rotational motion of the aryl subunit around the bond connecting this moiety to the meso-position of the BODIPY core. The energy barriers related to this rotation were simulated by DFT calculations. As follows from the relatively low rotational barrier calculated to about 17 kcal/mol, a free rotation is only possible for sterically unhindered compounds. Rotational barriers of more than 40 kcal/mol determined for the sterically hindered compounds suggest an effective freezing of the rotational motion in These molecules. With the aid of temperature-dependent spectroscopic measurements, we could show that the ability to rotate directly affects the optical properties of our set of BODIPY dyes. This accounts for the strong temperature dependence of the fluorescence of the sterically unhindered compounds which show a drastic decrease in fluorescence quantum yield and a significant shortening in fluorescence lifetime upon heating. The optical properties of the sterically hindered compounds, however, are barely affected by temperature. Our results suggest a nonradiative deactivation of the first excited singlet state of the sterically unhindered compounds caused by a conical intersection of the potential energy surfaces of the Ground and first excited state which is accessible by rotation of the meso-subunit. This is in good agreement with previously reported deactivation mechanisms. In addition, our results suggest the presence of a second nonradiative depopulation pathway of the first excited singlet state which is particularly relevant for the sterically hindered compounds.
Singlet oxygen can severely damage biological tissue, which is exploited in photodynamic therapy (PDT). In PDT, the effective range is limited by the distribution of the photosensitizer (PS) and the illuminated area. However, no distinction is made between healthy and pathological tissue, which can cause undesired damage. This encouraged us to exploit the more acidic pH of cancerous tissue and design pH-controllable singlet oxygen-generating boron-dipyrromethene (BODIPY) dyes. A pH sensitivity of the dyes is achieved by the introduction of an electronically decoupled, photoinduced electron transfer (PET)-capable subunit in meso-position of the BODIPY core. To favor triplet-state formation as required for singlet Oxygen generation, iodine substituents were introduced at the chromophore core. The resulting pH-controlled singlet oxygen-generating dyes with pKa values in the physiological range were subsequently assessed regarding their potential as pH-controlled PS for PDT.
Using HeLa cells, we could successfully demonstrate markedly different pH-dependent cytotoxicities upon illumination.
In summary, we report a highly modular solid TTA-UC system comprising of a crystalline, thermally stable PCN222(Pd) MOF with CA-coated MOF channels and with a DPA annihilator embedded in a solution-like environment in the MOF channels. This solid material displays blue upconverted delayed emission with a luminescence lifetime of 373 us, a threshold value of 329 mW*cm-2 and a triplet–triplet energy transfer efficiency of 82%. This optical application adds another facet to the versatile chemistry of PCN-222 MOFs. The design concept is also applicable to other TTA-UC pairs and enables tuning of the UCL color, for example, by replacing DPA with other dyes as exemplarily shown for 2,5,8,11-tetra-tert-butyl-perylene, that yields UCL at 450 nm. Current work aims to reduce the oxygen sensitivity and to increase the retention of the trapped annihilators in organic environments, for example, by tuning the chain length of the carboxylic acid and by coating the MOF surface. In addition, the TTA-UC efficiency will be further enhanced by reducing the reabsorption of the UC emission caused by Pd(TCPP) and by optimizing the sensitizer/annihilator interface.
The comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters require accurate and quantitative photo-luminescence measurements. This is of special importance for all photoluminescence applications in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
Bioanalytical, diagnostic, and security applications require the fast and sensitive determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats.[1,2] Ideal candidates for spectral encoding and multiplexing schemes are luminescent nanocrystals like semiconductor quantum dots (QDs), particularly Cd-containing II/VI QDs with their narrow and symmetric emission bands. With the availability of relatively simple and inexpensive instrumentation for time-resolved fluorescence measurements, similar strategies utilizing the compound-specific parameter fluorescence lifetime or fluorescence decay kinetics become increasingly attractive.[3-5] The potential of different types of QDs like II/VI, III/V and Cd-free ternary QDs such as AgInS (AIS) QDs for lifetime-based encoding and multiplexing has been, however, barely utilized, although the lifetimes of these nanocrystals cover a time windows which is barely accessible with other fluorophores. Here we present a brief insight into the photophysics of AIS QDs and show the potential of dye- and QD-encoded beads for lifetime-based encoding and detection schemes in conjunction with flow cytometry and fluorescence lifetime imaging microscopy
Optical spectroscopy – Techniques, instrumentation, and typical molecular and nanoscale reporters
(2018)
Different types of optical spectroscopies are introduced with special emphasis on method-inherent limitations and reliable instrument calibration and performance validation. In addition, different classes of molecular and nanocrystalline emitters are presented and the underlying photophysical processes are briefly described.
Different types of optical spectroscopies are introduced with special emphasis on method-inherent limitations and reliable instrument calibration and performance validation. In addition, procedures for the determination of spectroscopic key parameters like the photoluminescence quantum yield are presented including required instrument calibrations and material-specific effects related to certain emitters.
Mandatory for the comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters are reliable and quantitative photoluminescence measurements. This is of special relevance for all fluorescence applications in the life and material sciences. In the following, procedures for the determination of this spectroscopic key parameter are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
Relative and Absolute Methods for Measuring Photoluminescence Quantum Yields of UV/vis/NIR Emitters
(2019)
One of the key spectroscopic performance parameters of molecular and particulate emitters is the photoluminescence quantum yield (PL QY) that provides a direct measure for the number of emitted per absorbed photons. This triggered the interest in methods suitable for measuring this property for emitters in various environments in the UV/vis/NIR and above 1000 nm as well as on the ensemble and single emitter level. Moreover, for nonlinear emitters like lanthanide-based upconversion nanocrystals methods including instrumentation for power density-dependent PL QY studies are required.
An overview of the research activities in Division Biophotonics of BAM is given and suitable relative and absolute methods for the deter-mination of PL QY of organic dyes and different types of application-relevant nanomaterials in dispersion and in the solid state are presen-ted. This covers also the design and calibration of integrating sphere setups, achievable uncertainties, and candidates for PL QY reference materials.
The implementation of fluorescent methods is of outstanding importance in the field of optical chemical sensor Technology and biosciences. Their bioanalytical applications are manifold including fluorescence microscopy, fluorescence in situ hybridization, DNA sequencing, fluorescence-activated cell sorting, immunoassays, analysis of DNA and Protein microarrays, and quantitative PCR, just to name a few examples. Particularly, fluorescence microscopy is a valuable method in the versatile field of biomedical imaging methods which nowadays utilizes different fluorescence Parameters like emission wavelength/Color and lifetime for the discrimination between different targets. Sectional Images are available with confocal microscopes. Tissue, cells or single cellular compartments can be stained and visualized with fluorescent dyes and biomolecules can be selectively labeled with fluorescent dyes to Monitor biomolecular interactions inside cells or at Membrane bound receptors.
On the other hand , fluorophores can act as indicator (or "molecular probe") to visualize intrinsically colorless and non-fluorescent ionic and neutral analytes such as pH, Oxygen (pO2), metal ions, anions, hydrogen peroxide or bioactive small organic molecules such as Sugars or nucleotides. Thereby, their photoluminescent properties (fluorescence or phoporescence intensity, exitation and/or Emission wavelength, emission lifetime or anisotropy) respond to the presence of these species in their immediate Environment. In general, the use of luminescent probes has the advantage that they can be delivered directly into the sample, and detected in a contactless remote mode. By now, these probes are often encapsulated in different types of nanoparticles (NPs) made from (biodegradable) organic polymers, biopolymers or inorganic materials like silica or bound to their surface.