Liposome encapsulation of fluorescent nanoparticles: Quantum dots and silica nanoparticles
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Fluorescence Correlation Spectroscopy
Spatial fluorescence cross-correlation spectroscopy is a rarely investigated version of fluorescence correlation spectroscopy, in which the fluorescence signals from different observation volumes are cross-correlated. In the reported experiments, two observation volumes, typically shifted by a few microm, are produced, with a spatial light modulator and two adjustable pinholes. We illustrated the feasibility and potentiality of this technique by: i) measuring molecular flows, in the range 0.2-1.5 microm/ms, of solutions seeded with fluorescent nanobeads or rhodamine molecules (simulating active transport phenomenons); ii) investigating the permeability of the phospholipidic membrane of giant unilamellar vesicles versus hydrophilic or hydrophobic molecules (in that case the laser spots were set on both sides of the membrane). Theoretical descriptions are proposed together with a discussion about fluorescence-correlation-spectroscopy-based, alternative methods.
Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
Spatial light modulator
Rhodamine
Spatial correlation
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Fluorescence correlation spectroscopy reveals that an oligonucleotide, the T3 promoter primer, undergoes only lateral diffusion when adsorbed to the interface of water and silica chemically modified with a hydrocarbon. The autocorrelation decay fits well to the model of simple diffusion, reporting a diffusion coefficient of 1.8 × 10 −6 cm 2 /s. Single-molecule resolution of bursts for the T3 promoter primer reveals that rare, strong adsorption punctuates the lateral diffusion. Removal of the strong adsorption events from the data set, followed by autocorrelation, shows the actual diffusion coefficient to be 2.8 × 10 −6 cm 2 /s, which is comparable to other oligonucleotides of the same size at the same interface. The single-molecule measurements show that average duration of strong adsorption is 0.2 s, and the average fraction of strongly adsorbed molecules is 10% of the molecules at the interface. While single-molecule spectroscopy reveals a process not evident in fluorescence correlation spectroscopy, the precision of the parameters describing strong adsorption is limited by the statistics of small numbers. Fluorescence correlation spectroscopy is suited to observing a much larger number of events, which is needed for high precision. The two methods are complementary: single-molecule spectroscopy gives estimates of the chemical parameters needed for design of the fluorescence correlation spectroscopy, achieving precise measurements with an accurate interpretation.
Fluorescence Correlation Spectroscopy
Single-molecule experiment
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Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
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Ganglioside (GM1) micelles have been studied by means of three different techniques: fluorescence correlation spectroscopy (FCS), electronic energy transfer, as monitored by time-resolved fluorescence spectroscopy, as well as static and dynamic light scattering. The aggregation numbers obtained, 168 ± 4, remain constant over a wide range of GM1 concentrations (0.764–156 μM), are very consistent when using different donor–acceptor energy transfer pairs and have served as reference values in tests of the FCS method. It is recommended to calibrate the focal volume by using known dye concentrations. For this the rhodamine dye, 5-TAMRA, turns out to be most suitable. It is also shown that FCS provides correct values of the aggregation numbers, provided that the focal volume is calibrated by using updated values of the diffusion constant of Rhodamine 6G. These results also support recent methodological advances in FCS.
Fluorescence Correlation Spectroscopy
Rhodamine 6G
Acceptor
Rhodamine
Binding constant
Fluorescence spectrometry
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Fluorescence lifetime correlation spectroscopy (FLCS) is a recently developed method which combines the conventional fluorescence correlation spectroscopy (FCS) and time correlated single photon counting (TCSPC). It enables to perform a signal separation of the species which possess different lifetime. Particular diffusion components of a mixture of more fluorescent species can be thus separated. Moreover, the detector afterpulsing can be suppressed by FLCS.
Fluorescence Correlation Spectroscopy
Photon Counting
Fluorescence cross-correlation spectroscopy
SIGNAL (programming language)
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Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.
Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
Photon Counting
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Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
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A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate Kd from a single binding measurement.
Fluorescence Correlation Spectroscopy
Fluorescence spectrometry
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Thioflavin
Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
Amyloid (mycology)
Quantum yield
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Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
Saturation (graph theory)
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