Fluorescence steady-state detection

One chooses dye pairs such that Ro is similar in magnitude to the expected distance of interest. Calculation of dye-to-dye distances from the fluorescence steady-state data allow the structure of the probe-DNA assembly to be inferred. Dynamic FRET measurements can be used to infer the time scales of DNA motion (see below). A related strategy for hybridization detection is known as the molecular beacon strategy A single strand of DNA that folds up to form a hairpin structure is doubly labeled on S and 3 ends with donor and acceptor such that the donor does not emit due to close contact by the acceptor upon hybridization with its target complementary DNA, the labeled strand straightens out, the donor and acceptor move far apart, and the donor emission is observed. 

The detection limits in the table correspond generally to the concentration of an element required to give a net signal equal to three times the standard deviation of the noise (background) in accordance with lUPAC recommendations. Detection limits can be confusing when steady-state techniques such as flame atomic emission or absorption, and plasma atomic emission or fluorescence, which... 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

FIAs can be based on steady-state intensity measurements without probe amplification, owing to the sensitivity of detection that is possible with fluorescence instrumentation, which exceeds that of spectrophotometers by two or three orders of magnitude. A sensitive fluorometer has been described for an estradiol assay(36) in which the limit of estradiol detection is 3 x KT11 M. Estradiol antibody labeled with rhodamine B is reacted with estradiol samples. Unreacted labeled antibody is removed with Sepharose-estradiol-casein beads, and the remaining fluorescence is directly proportional to the analyte concentration. The detection limit of rhodamine B on the same fluorometer is 5 x 1(T12 M. This instrument uses a 0.75 mW green helium-neon (HeNe) laser to irradiate the sample from above, at the air-liquid interface, to increase the light path and to decrease surface reflections. The sample compartment has a top-mounted photon trap, and a mirror mounted on the side of the sample compartment opposite the PMT to enhance detection. 

Time-resolved luminescence spectroscopy may be extremely effective in minerals, many of which contain a large amount of emission centers simultaneously. With the steady state technique only the mostly intensive centers are detected, while the weaker ones remain unnoticed. Fluorescence in minerals is observed over time range of nanoseconds to milliseconds (Table 1.3) and this property was used in our research. Thus our main improvement is laser-induced time-resolved spectroscopy in the wide spectral range from 270 to 1,500 nm, which enables us to reveal new luminescence centers in minerals previously hidden by more intensive centers. 

The emitted light is detected along y through a polarizer oriented either along z (Fz) or along x (Fx). In fluorescence polarization studies with continuous excitation (steady-state experiments), the emission anisotropy r and the emission polarization p are defined in eqs 8a and 8b. 

The last term is a small correction (< 5%) and is probably beyond experimental detection. As more quenching of fluorescence occurs by quenchers which were in excess of the steady-state concentration, the departures from the Stern—Volmer limiting law becomes more marked. Nevertheless, determining the slope of T0/T([ Q]) versus the quencher concentration and the lifetime of the excited state of the fluorophor in... 

Steady-state measurements of the fluorescence anisotropy of fluorescein derivatives form the basis of a sensitive analytical technique also used to detect and quantitate proteins [36], steroids [37-39], therapeutic drugs, and narcotics [40-42], In a different approach, the anisotropy of the fluorescein conjugate is measured as a function of the medium viscosity to determine the segmental mobility of the chains that cover the binding site [43-45],... 

Increasing the solvent polarity results in a red shift in the -t -amine exciplex fluorescence and a decrease in its lifetime and intensity (113), no fluorescence being detected in solvents more polar than tetrahydrofuran (e = 7.6). The decrease in fluorescence intensity is accompanied by ionic dissociation to yield the t-17 and the R3N" free radical ions (116) and proton transfer leading to product formation (see Section IV-B). The formation and decay of t-17 have been investigated by means of time resolved resonance Raman (TR ) spectroscopy (116). Both the TR spectrum and its excitation spectrum are similar to those obtained under steady state conditions. The initial yield of t-1 is dependent upon the amine structure due to competition between ionic dissociation and other radical ion pair processes (proton transfer, intersystem crossing, and quenching by ground state amine), which are dependent upon amine structure. However, the second order decay of t-1" is independent of amine structure... 

The steady-state fluorescence measurements of pyrene in supercritical CO2 were made with a spectrometer assembly consisting mainly of Kratos optical parts. The custom built high pressure optical cell is equipped for detection at 90°. The emission was detected with a Hammamatsu IP-28 photomultiplier tube. The... 

Experimentally, commercial steady-state fluorescence spectrometers can be equipped with polarizer attachments, either sheet or Glan-Thompson polarizers. Alternatively, sheet polarizers are usually easily incorporated into the sample cavity in the excitation and emission pathways. Likewise, for time-resolved spectroscopy, polarizers may simply be introduced into the excitation and detection paths. Frequently, the excitation source in time-resolved experiments is a laser which will be inherently polarized. 

Laser-induced fluorescence is a sensitive, spatially resolved technique for the detection and measurement of a variety of flame radicals. In order to obtain accurate number densities from such measurements, the observed excited state population must be related to total species population therefore the population distribution produced by the exciting laser radiation must be accurately predicted. At high laser intensities, the fluorescence signal saturates (1, 2, 3 ) and the population distribution in molecules becomes independent of laser intensity and much less dependent on the quenching atmosphere (4). Even at saturation, however, the steady state distribution is dependent on the ratio of the electronic quenching to rotational relaxation rates (4, 5, 6, 7). When steady state is not established, the distribution is a complicated function of state-to-state transfer rates. 

Method 3. Saturation Method With Peak Detection. In this method, developed by (imenetto and Winefordner2,3, it is necessary to excite fluorescence 3+1 with 1+3 and a short time later (< 1 ys) excite 3+1 with 2+3. In this case the atomic system effectively acts on a 2-level atom since excitation and measurement of fluorescence is done at the peak of the excitation pro-fil prior to relaxation of the system to a 3-level steady state process The temperature here is related simply to the ratio BP /Bp and statistical weights of the levels and is independ-r3+l... 

In good solvents at ambient temperature, the excited state (67 ) will quickly relax to the planar form, so that only the 0-0 emission from Si is detected in steady-state emission. If the same experiment is performed at low temperature and in a viscous solvent, the molecular torsion of 67 in attaining its planar form is hampered by the medium, and planarization is slow on the timescale of the fluorescence lifetime (355 ps). Emission will not only occur from the potential minimum of the lowest excited state, but from virtually all frozen ro-tamers resulting in a broad and blue-shifted spectrum. Only after planarization of 67 is complete narrow emission from the lowest excited-state conformation will reoccur. Consequently, planarization of the excited state rather than energy migration is likely to govern the emission behavior in PPEs such as 12. 

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