Academic literature on the topic 'Fluorophore Quenching'

The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon—called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 μM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.

Covalent linkage of fluorophores and photostabilizers was recently revived as a strategy to make organic fluorophores “self-healing” via triplet-state quenching. Although Lüttke and co-workers pioneered this strategy already in the 1980s, the general design principles still remain elusive. In this contribution, we combine experiments and theory to understand what determines the photostabilization efficiency in dye–photostabilizer conjugates. Our results from single-molecule microscopy and molecular dynamics simulations of different Cy5-derivatives suggest that the distance and relative geometry between the fluorophore and photostabilizer are more important than the chemical nature of the photostabilizer, e.g. its redox potential, which is known to influence electron-transfer rates. We hypothesize that the efficiency of photostabilization scales directly with the contact rate of the fluorophore and photostabilizer. This study represents an important step in the understanding of the molecular mechanism of intramolecular photostabilization and can pave the way for further development of stable emitters for various applications.

The quenching of the fluorescence of five aromatic hydrocarbons by three halogenated organics and by molecular oxygen has been measured. Both fluorescence intensity and fluorescence lifetime measurements have been employed to validate results and interpretation; linear Stern–Volmer analyses are shown to apply throughout. The fluorescence quenching rate constant of molecular oxygen for the five aromatic hydrocarbons is essentially equivalent to the diffusion rate constant independent of the fluorophore excitation energy. The halogenated organic–fluorophore rate constants vary by a factor of 965 and are shown to correlate roughly with the energy difference between the quencher and fluorophore excited electronic states in accord with a standard model of quantum two-level mixing. The value of the coupling interaction energy is ∼2500 cm−1.

A series of water-soluble 1-amino-naphthalenes and 2-amino-fluorenes are prepared. These serve as model fluorophores for measuring the thermodynamics and kinetics of fluorescence quenching with phenylboronic acids and aliphatic amines. Steady-state and time-resolved fluorescence quenching kinetics are investigated using the Stern–Volmer method. Diffusion limited quenching constants and exergonic thermodynamics of electron transfer are derived for the 5-amino-1-napthol and 2-aminofluorene derivatives with phenylboronic acid and/or an aliphatic imine. No quenching and endergonic thermodynamics or electron transfer are observed for 5-sulfonamide, 5-sulfonic acid, or 5-hydroxy-7-sulfonic acid aminonaphthalene derivatives. Boronic acid sensors synthesized from these aminofluorophores by reductive amination with 2-formylphenylboronic acid undergo fluorescence revival in the presence of saccharides only when the fluorophore demonstrates diffusion limited quenching kinetics and exergonic thermodynamics of electron transfer with the boronic acid or imine quenchers. Thus, these two properties are suitable empirical tools for predicting saccharide-induced fluorescence revival of boronic acid sensors.

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscoopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and α-actinin bands of skeletal muscle tissue and also actin and α-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM. (J Histochem Cytochem 55: 983–990, 2007)

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3′,6′-di( O-thiosalicyl)fluorescein, SSP4), and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n = 1–7) and H2S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

Metal nanoparticles have recently gained popularity in many research areas due to their nanosize-related properties. Depending on the size of the metal nanoparticle, their mode of interaction with electromagnetic radiation and the outcome of this interaction vary; in turn the effect exerted on a protein which is conjugated to a nanoparticle varies, because different sized nanoparticles demonstrate different modes of energy transfer with electromagnetic radiation and molecules conjugated to them. Very small cluster with sizes around 1 – 1.2 nm tend to get excited by incident light and emit fluorescence, whereas larger nanoparticles absorb the incoming light very strongly due to their LSPR. In this study we observed the outcomes of the interaction between two types of nanoparticles, namely gold and gold/silver alloyed nanoparticles with the fluorescence emission of two fluorophores, namely eGFP and rPhiYFP; and demonstrated a bioassay where the fluorescence modulation by gold nanoparticles can be used as the sensing strategy. Lastly, we demonstrated the potential of autofluorescent gold nanoparticles as intracellular reporters.

For determination of structures and structural dynamics of proteins organic fluorophores are a standard instrument. Intra- and intermolecular contact of biomolecular structures are determined in time-resolved and stationary fluorescence microscopy experiments by quenching of organic fluorophores due to Photoinduced Electron Transfer (PET) and dimerization interactions. Using PET we show in this work that end-to-end contact dynamics of serine-glycine peptides are slowed down by glycosylation. This slow down is due to a change in reaction enthalpy for end-to-end contact and is partly compensated by entropic effects. In a second step we test how dimerization of MR121 fluorophore pairs reports on end-to-end contact dynamics. We show that in aqueous solutions containing strong denaturants MR121 dimerization reports advantageously on contact dynamics for glycine-serine oligopeptides compared to the previously used MR121/tryptophane PET reporters. Then we analyze dimer interactions and quenching properties of different commercially available fluorophores being standards in Förster Resonance Energy Transfer (FRET) measurements. Distances in biomolecules are determinable using FRET, but for very flexible biomolecules the analysis of masurement data can be distorted if contact of the two FRET fluorophores is likely. We quantify how strong the quenching of fluorophore pairs with two different or two identical fluorophores is. Dimer spectra and association constants are quantified to estimate if fluophores are applicable in various applications, e.g. in FRET measurements with unstructured peptides and proteins
Zur Charakterisierung von Proteinen werden in der fluoreszenzbasierten Mikroskopie organische Farbstoffe benutzt, um strukturelle Informationen bzw. Informationen über dynamische Prozesse zu gewinnen. In der zeitaufgelösten und stationären Fluoreszenzmikroskopie können hiermit Kontaktprozesse durch photoinduzierten Elektronentransfer und auch Dimerisierung der Fluorophore analysiert werden. In dieser Arbeit wird mittels photoinduziertem Elektronentransfer PET gezeigt, dass Glykosylierung End-zu-End Kontaktkinetiken verändert. Sehr flexible Serin-Glycin Peptide zeigen glykosyliert langsamere Kinetiken durch Veränderung der Reaktionsenthalpie der Kontaktreaktion beider Peptidenden verglichen zu unglykosylierten. Diese enthalpischen Beiträge werden zum Teil von entropischen Beiträgen kompensiert. Außerdem wird gezeigt, dass Glycin-Serin Peptiddynamiken auch mittels Farbstoffpaaren gemessen werden können, die auf Löschwechselwirkungen durch Dimerisierung beruhen. Die Stärke dieser Löschwechselwirkungen hängt vom Farbstoffpaar ab. In Lösungen mit Denaturierungsmitteln können Farbstoffpaare des Fluoreszenzfarbstoffes MR121 vorteilhaft für Messungen von Dynamiken von Glycin-Serin Peptiden genutzt werden. Die Dimerwechselwirkungen können bei sehr flexiblen Biomolekülen und möglichem Kontakt von Fluorophoren die konventionelle Analyse von Förster Resonanz Energie Transfer (FRET) Messungen erschweren. Wir untersuchen an Glycin-Serin Oligopeptiden das Dimerisierungsverhalten kommerziell erhältlicher Fluorophore, die in FRET Messungen verwendet werden. Für gleiche und verschiedene Fluorophore wird die Löschung durch Dimerwechselwirkungen quantifiziert. Dabei werden Dimerspektren und Assoziationskonstanten für Dimerisierungsreaktionen bestimmt. Letztere helfen bei der Abschätzung, ob Fluorophorpaare für verschiedene Anwendungen geeignet sind, zum Beispiel in FRET-Messungen in unstrukturierten Peptiden und Proteinen

Les propriétés intrinsèques, photophysiques, électrochimiques et cristallographiques des molécules fluorescentes 4,4'-bis(2-benzoxazolyle)stilbène (BBS) et 2,5-bis(5-tert-butyl-2-benzoxazolyle)thiophène (BBT) ont été étudiées en solution et dans les polymères semi-cristallins : poly(butylène succinate) (PBS) et polylactide (PLA). Les deux fluorophores sont caractérisés par de hauts rendements quantiques absolus de fluorescence. Toutefois, une désactivation de la fluorescence peut se produire par croisement intersystème vers l'état triplet pour le BBT, et par photoisomérisation trans-cis pour le BBS. La cinétique de ce dernier processus dépend de la concentration, résultant en un pur isomère cis photo-induit à faibles concentrations, qui est accompagné à des concentrations élevées par l'apparition d'un composé acide après photo-clivage suivi d'une oxydation. Cette étude a révélé des changements spectroscopiques prononcés suite à l’augmentation de la concentration des fluorophores, en particulier à l'état solide, spécifiques à l'agrégation des molécules à l'état fondamental pour le BBT et à la formation d’excimères pour le BBS, permettant ainsi de corréler les propriétés fluorescentes avec les caractéristiques du monocristal pour chaque fluorophore. En outre, le passage d’une dispersion moléculaire à une séparation de phases dans le cas du BBS est accompagné d'un changement de couleur du bleu au vert, qui est sensible à la déformation, à la température et au temps, affectant les rendements quantiques absolus de fluorescence et fournissant une large opportunité à la création d'une grande variété de polymères intelligents indicateurs capables d'auto-évaluation. D’autre part, la solubilité élevée du BBT dans les solvants courants, combinée à ses propriétés optoélectroniques élevées, en font un candidat en tant que référence universelle de fluorescence et matériau intelligent à la fois pour les études de polymères et en solution. Similairement aux mélanges comprenant des polymères miscibles, l'orientation du PBS augmente après ajout d'une molécule fluorescente, dont les monomères ont tendance à être orientés dans des films étirés, contrairement aux excimères ou agrégats.
The intrinsic, photophysical, electrochemical and crystallographic properties of the fluorescent molecules 4,4'-bis(2-benzoxazolyl)stilbene (BBS) and 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene (BBT) have been investigated in solution as well as in semi-crystalline poly(butylene succinate) (PBS) and polylactide (PLA). Both fluorophores were found to be characterized by high fluorescence absolute quantum yields. However, fluorescence quenching was found to occur by intersystem crossing to the triplet state for BBT, and by trans-cis photoisomerization for BBS. The latter process was kinetically concentration-dependent, resulting in a pure photoinduced cis-isomer at low concentrations, which is accompanied at high concentrations by the appearance of an acidic compound after photocleavage followed by oxidation. This study revealed pronounced spectroscopic changes upon an increase of the fluorophore concentration, especially in the solid-state, specific to aggregation of ground state molecules for BBT or excimer formation for BBS, thus correlating the fluorescent features with the single crystal characteristics for each fluorophore. Moreover, the transformation from molecular dispersion to phase separation, occurring for BBS, is accompanied by a significant colour change from blue to green, which is sensitive to deformation, time and temperature, thus affecting the absolute fluorescence quantum yields and providing a broad opportunity for the creation of a wide variety of smart polymers with self-assessing capabilities. On the other hand, the BBT’s high solubility in common solvents combined with its measured enhanced optoelectronic properties make it a candidate as a universal fluorophore reference and smart material for both polymeric and solution studies. Similarly to blends comprising miscible polymers, the PBS orientation was found to increase by adding a fluorescent molecule, whose monomers tend to be oriented in stretched films, contrary to excimers or aggregates.