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- Fluorescence (9)
- Multiplexing (7)
- Quantum dot (6)
- Aggregation (4)
- Lifetime (4)
- Quantum yield (4)
- Dye (3)
- FRET (3)
- Photoluminescence (3)
- Assay (2)
Bright, long-lived emission from first-row transition-metal complexes is very challenging to achieve. Herein, we present a new strategy relying on the rational tuning of energy levels. With the aid of the large N-Cr-N bite angle of the tridentate ligand ddpd (N,N′-dimethyl-N,N′-dipyridine-2-ylpyridine-2,6-diamine) and its strong σ-donating capabilities, a very large ligand-field splitting could be introduced in the chromium(III) complex [Cr(ddpd)2]3+, that shifts the deactivating and photoreactive 4T2 state well above the emitting 2E state. Prevention of back-intersystem crossing from the 2E to the 4T2 state enables exceptionally high near-infrared phosphorescence quantum yields and lifetimes for this 3d metal complex. The complex [Cr(ddpd)2](BF4)3 is highly water-soluble and very stable towards thermal and photo-induced substitution reactions and can be used for fluorescence intensity- and lifetime-based oxygen sensing in the NIR.
Acoustic levitation is used as a newtool to study concentration-dependent processes influorescence
spectroscopy. With this technique, small amounts of liquid and solid samples can be measured
without the need for sample supports or containers, which often limits signal acquisition and
can even alter sample properties due to interactions with the support material. We demonstrate
that, because of the small sample volume, fluorescence measurements at high concentrations
of an organic dye are possible without the limitation of inner-filter effects, which hamper such
experiments in conventional, cuvette-based measurements. Furthermore, we show that acoustic
levitation of liquid samples provides an experimentally simple way to study distance-dependent
fluorescence modulations in semiconductor nanocrystals. The evaporation of the solvent during
levitation leads to a continuous increase of solute concentration and can easily be monitored by
laser-induced fluorescence.
We systematically assessed the loading behavior of core-multishell nanoparticles (CMS NPs) for the solvatochromic dyes Coumarin 153 and Nile Red and studied the influence of the guest and its concentration on CMS NP aggregation using steady state absorption and fluorescence spectroscopy and dynamic light scattering (DLS). These measurements revealed the strong fluorescence of dye-loaded CMS NPs and formation of nonemissive dye aggregates in the outer CMS layer at higher loading concentrations of Nile Red, whereas in the case of Coumarin 153, a new species with red-shifted absorption and blue-shifted emission appeared. Moreover, dye loading triggers an aggregation of CMS NPs which have a hydrodynamic radius of 8 nm, thereby leading to CMS aggregates with a radius of 100–120 nm. These results underline the need for systematic studies of the influence of the guest and its loading concentration on CMS NP size for cellular uptake and in vivo imaging studies and the rational design of CMS NPs with improved transport and targeting abilities.
In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Förster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies. Methods: We coupled the fluorophores DY-682 (donor; absorption [abs]/emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features. Results: When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgGDY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice. Conclusion: Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
The photoluminescence quantum yield (Φf) that presents a direct measure for the efficiency of the conversion of absorbed photons into emitted photons is one of the spectroscopic key parameters of functional fluorophores. It determines the suitability of such materials for applications in, for example, (bio)analysis, biosensing, and fluorescence imaging as well as as active components in optical devices. The reborn interest in accurate Φf measurements in conjunction with the controversial reliability of reported Φf values of many common organic dyes encouraged us to compare two relative and one absolute fluorometric method for the determination of the fluorescence quantum yields of quinine sulfate dihydrate, coumarin 153, fluorescein, rhodamine 6G, and rhodamine 101. The relative methods include the use of a chain of Φf transfer standards consisting of several 'standard dye' versus 'reference dye' pairs linked to a golden Φf standard that covers the ultraviolet and visible spectral region, and the use of different excitation wavelengths for standard and sample, respectively. Based upon these measurements and the calibration of the instruments employed, complete uncertainty budgets for the resulting Φf values are derived for each method, thereby providing evaluated standard operation procedures for Φf measurements and, simultaneously, a set of assessed Φf standards.
To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1−4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.
Dye–biomolecule conjugation is frequently accompanied by considerable spectral changes of the dyes absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.
Photo-initiated cross-linking of multifunctional acrylic esters in polymeric binders was investigated based on digital imaging using the Computer-to-Plate (CtP) technology applying laser exposure in the near-infrared (NIR). Generation of initiating radicals occurs by electron transfer from the excited state of the NIR-sensitizer to the radical generator, an onium salt. Iodonium salts derived from several borates and those with the bis(trifluoromethylsulfonyl)imide anion resulted in lithographic materials with high sensitivity. Photo-induced electron transfer plays a major function to generate initiating radicals by a sensitized mechanism but thermal events also influence sensitivity of the coating. Internal conversion was the major deactivation pathway while a certain fraction of NIR-dye fluorescence was also available. A line shape focused laser system with emission in the NIR was successfully used to bake the materials.
The synthesis and characterization of the new dinuclear dipeptide [(EtOOC-tpy)Ru(tpy-NHCO-tpy)Ru(tpy-NHCOCH3)]4+ 34+ of the bis(terpyridine)ruthenium amino acid [(HOOC-tpy)Ru(tpy-NH2)]2+ 12+ are described, and the properties of the dipeptide are compared to those of the mononuclear complex [(EtOOC-tpy)Ru(tpy-NHCOCH3)]2+ 42+ carrying the same functional groups. 34+ is designed to serve a high electronic similarity of the two ruthenium sites despite the intrinsic asymmetry arising from the amide bridge. This is confirmed via UV–vis absorption and NMR spectroscopy as well as cyclic voltammetry. 42+ and 34+ are emissive at room temperature, as expected. Moreover, 34+ exhibits dual emission from two different triplet states with different energies and lifetimes at room temperature. This is ascribed to the presence of a unique thermal equilibrium between coexisting [RuII(tpy-NHCO-tpy·)RuIII] and [RuIII(tpy-NHCO-tpy·)RuII] states leading to an unprecedented excited-state RuIIRuIII mixed-valent system via the radical anion bridge tpy-NHCO-tpy·. The mixed-valent cation 35+, on the other hand, shows no measurable interaction of the RuIIRuIII centers via the neutral bridge tpy-NHCO-tpy (Robin–Day class I). Reduction of 34+ to the radical cation 33+ by decamethylcobaltocene is bridge-centered as evidenced by rapid-freeze electron paramagnetic resonance spectroscopy. Interestingly, all attempts to observe 33+ via NMR and UV–vis absorption spectroscopy only led to the detection of the diamagnetic complex 3-H3+ in which the bridging amide is deprotonated. Hence 3-H3+ (and 4-H+) appear to reduce protons to dihydrogen. The ease of single and double deprotonation of 42+ and 34+ to 4-H+, 3-H3+, and 3-2H2+ was demonstrated using a strong base and was studied using NMR and UV–vis absorption spectroscopies. The equilibrating excited triplet states of 34+ are reductively quenched by N,N-dimethylaniline assisted by hydrogen bonding to the bridging amide.