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Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and...
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Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.
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This chapter presents a concise introduction into the method of Fluorescence Lifetime Correlation Spectroscopy (FLCS). This is an extension of Fluorescence Correlation Spectroscopy (FCS) that analyses fluorescence intensity fluctu...
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This chapter presents a concise introduction into the method of Fluorescence Lifetime Correlation Spectroscopy (FLCS). This is an extension of Fluorescence Correlation Spectroscopy (FCS) that analyses fluorescence intensity fluctuations from small detection volumes in samples of ultra-low concentration. FCS has been widely used for investigating diffusion, conformational changes, molecular binding/unbinding equilibria, or chemical reaction kinetics, at single molecule sensitivity. In FCS, this is done by calculating intensity correlation curves for the measured intensity fluctuations. FLCS extends this idea by calculating fluorescence-lifetime specific intensity correlation curves. Thus, FLCS is the method of choice for all studies where a parameter of interest (conformational state, spatial position, molecular environmental condition) is connected with a change in the fluorescence lifetime. After presenting the theoretical and experimental basis of FLCS, the chapter gives an overview of its various applications. (C) 2018 The Authors. Published by Elsevier Inc.
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Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of
fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented
their widespread...
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Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of
fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented
their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested
as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged
system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal
optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing
in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining
its resolution. We demonstrate a 4×4×18 cm3 sized fluorescence correlation spectrometer, which requires no
alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much
larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by
the large background fluorescence of the commonly available optical fibers. We propose this as a portable and
field deployable single molecule device with practical diagnostic applications.
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Cross-reactivity and multispecific functionality of antibodies play a central role in the immune system. The Ab's promiscuity is attributed to structural flexibility and conformational multiplicity of its binding sites governed by...
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Cross-reactivity and multispecific functionality of antibodies play a central role in the immune system. The Ab's promiscuity is attributed to structural flexibility and conformational multiplicity of its binding sites governed by the rearrangement of hydrogen bonding centers. However, antibodies whose recognition and binding rely on less directional hydrophobic interactions might follow different interaction pathways. We investigated interaction of anti-polycyclic aromatic hydrocarbon mAb with two biologically important cross-reactants, pyrene and benzo(a)pyrene. Complex formation was characterized by means of low-temperature laser-induced fluorescence spectroscopy in both low- and high-resolution fluorescence line-narrowing (FLN) modes. It is shown that the FLN spectroscopy can identify various haptens cross-reacted with an Ab, as well as simultaneously differentiate between free and immunocomplexed haptens. In addition, our results suggest an interesting case of an Ab binding a particular cross-reactant by adopting two distinct conformations of its binding sites. The existence of the multiple conformations for anti-polycyclic aromatic hydrocarbon mAb that are trapped at low temperature can be rationalized through the existing models for Ab binding. Finally, as revealed by FLN spectra of immunocomplexed chromophores, π-π interactions, rather than hydrogen bonding, play the central role in complex formation.
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A spectrofluorometer capable of dispersed-spectrum, simultaneous, multiwavelength UV excitation and collection of luminescence has been constructed for the purpose of qualitatively and quantitatively determining aromatic hydrocarb...
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A spectrofluorometer capable of dispersed-spectrum, simultaneous, multiwavelength UV excitation and collection of luminescence has been constructed for the purpose of qualitatively and quantitatively determining aromatic hydrocarbon pollutants dissolved in ocean water, Hydrocarbon fluorescence data produced by this instrument were in the form of excitation/emission matrices, which provide more spectral information about these complex mixtures than is available from conventional excitation or emission fluorescence profiles. Second-order statistical methods were applied to these data to determine low part-per-billion concentrations of two primary fluorescent compounds, naphthalene and styrene, found in ocean water exposed to gasoline despite the presence of uncalibrated interference from similar aromatic compounds, the ocean water matrix, and the instrumental background.
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Fluorescence imaging is a powerful technique for visualizing temporal and spatial changes of biological events in living samples by utilizing fluorescent probes whose properties are altered upon interaction with the target biomole...
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Fluorescence imaging is a powerful technique for visualizing temporal and spatial changes of biological events in living samples by utilizing fluorescent probes whose properties are altered upon interaction with the target biomolecule. In the past few years, many fluorescent probes have been developed based on xanthene dyes in which the O atom at the 10-position is replaced with a Si atom, such as 2,7-bis(dimethylamino)9,9-dimethyl- 9-sila-9H-anthracenium (TMDHS), Si-rhodamines and TokyoMagentas. Interestingly, these fluorophores not only retain the advantages of the typical xanthene dyes, but also have a number of unique properties, including far-red to nearinfrared emission. This review summarizes silicon-substituted xanthene dyes reported to date, along with their photophysical properties. We include examples of applications of probes utilizing these fluorophores, such as multi-color imaging and super-resolution live-cell microscopy.
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We investigate the time evolution of the excitonic polarization in semiconducting carbon nanotubes by a quantum theory, and present a modulation scheme of the excitonic polarization by applying a weak laser field. In calculation, ...
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We investigate the time evolution of the excitonic polarization in semiconducting carbon nanotubes by a quantum theory, and present a modulation scheme of the excitonic polarization by applying a weak laser field. In calculation, we consider the many-body interaction between the electrons and the holes. Our results indicate that there is an intermittency of excitonic polarization. A correlation effect of the excitons induced by the weak laser field may be responsible for this phenomenon. We discuss the effect of the nanotube radius, laser field strength, and laser field frequency on the excitonic polarization.
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Small changes of local pH in the vicinity of an electrode surface are observed and quantified for the first time using the infrared attenuated total reflection technique (ATR). The local pH changes occur during hydrogen evolution ...
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Small changes of local pH in the vicinity of an electrode surface are observed and quantified for the first time using the infrared attenuated total reflection technique (ATR). The local pH changes occur during hydrogen evolution close to the ATR prism used as the cathode in an electrochemical cell containing a phosphate buffered electrolyte. pH changes are deduced directly from the phosphate concentration changes. Under optimum conditions, a change as small as 10(-4) pH unit can be measured. A linear approximation model of the transport in the cell accounts very well for the dependence of pH changes on the electrochemical current density, but fails to give an absolute prediction as accurate as the experimental determination. These observations offer new possibilities of measuring small changes of local pH in a wide pH range using different acid-base pairs. (C) 1997 Eisevier Science S.A. [References: 10]
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Aim. Here we synthesize a series of benzoindolium squaraine dyes with N-substituents (SQ) andexamine these dyes as fluorescent probes to detect serum albumins. Methods. Organic synthesis,fluorescent spectroscopy, absorption spectr...
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Aim. Here we synthesize a series of benzoindolium squaraine dyes with N-substituents (SQ) andexamine these dyes as fluorescent probes to detect serum albumins. Methods. Organic synthesis,fluorescent spectroscopy, absorption spectroscopy, fluorescent microscopy. Results. The spectralluminescent properties of SQ dyes in the aqueous solution were investigated in the presence ofbovine serum albumin (BSA), human serum albumin (HSA), equine serum albumin (ESA),ovalbumin (OVA), beta-lactoglobulin (BLG) and lysozyme (LYS), as well as in the presence ofnucleic acid. The maxima of excitation spectra of the studied dyes in the buffer solution rangedwithin 623–673 nm, with the fluorescence emission maxima lying between 635 and 690 nm. Allthese dyes showed a similar increase in the fluorescence intensity with serum albumins and at thesame time with the red shift up to 12 nm, which could point out the binding of the dyes to proteins.These dyes demonstrate a noticeably lower fluorescence intensity in the presence of OVA, BLG,and LYS, which structurally differ from serum albumins. The studied dyes gave no significantfluorescent response to the addition of nucleic acids. The equilibrium constants of dyes bindingto BSA (K) were estimated as 3.0 ± 0.3x105 M-1 for SL-2411 and 2.4 ± 0.6x105 M-1 for SL-2412.Based on the relative values of K, we could suppose that the mechanism of dye-BSA binding isthe interaction of the chromophore of the dye with the protein globule. It was shown that SL-2411penetrates the cell membrane and distributes in the cytoplasm without co-localization withMitoTracker Green. Conclusion. These dyes could apply fluorescent spectroscopy for proteindetection and potentially visualize cell components with minimum to no autofluorescence.
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Dextromethorphan (Figure 1) is commonly used as an antitussive, or cough suppressant (1). Unlike codeine (the most commonly used narcotic cough suppressant), dextromethorphan is available over-the-counter (OTC). The most common fo...
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Dextromethorphan (Figure 1) is commonly used as an antitussive, or cough suppressant (1). Unlike codeine (the most commonly used narcotic cough suppressant), dextromethorphan is available over-the-counter (OTC). The most common form has the IUPAC name 3-methoxy-17-methyl-(9α,13α,14α)-morphinan hydrobromide monohydrate and occurs as white crystals.
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