Fluorophores energy transfer

Fig. 46. Possible mechanism for chemiluminescence of oxalate esters. Attack by peroxide ions leads to a peroxyoxalate intermediate which, in turn, breaks down to an unstable excited-state dio-xetaneone. In the presence of an appropriate fluorophore, energy transfer from the dioxetaneone leads to luminescence from the fluorescent sensitizer.

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

The intercept, 1/Po, is called the anisotropy of the molecule and is an indication of the nonrotational depolarization of the molecule. This intrinsic depolarization is due to the segmental motion of the fluorophores within the molecule the depolarization due to energy transfer and the angular difference in transition dipole moments of the absorbing and emitting states. 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

As seen from (1) and (2), intermolecular processes may reduce essentially the lifetime and the fluorescence quantum yield. Hence, controlling the changes of these characteristics, we can monitor their occurrence and determine some characteristics of intermolecular reactions. Such processes can involve other particles, when they interact directly with the fluorophore (bimolecular reactions) or participate (as energy acceptors) in deactivation of S) state, owing to nonradiative or radiative energy transfer. Table 1 gives the main known intermolecular reactions and interactions, which can be divided into four groups ... 

Forde, T. and Hanley, Q. S. (2006). Spectrally resolved frequency domain analysis of multi-fluorophore systems undergoing energy transfer. Appl. Spectrosc. 60, 1442-52. 

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

Fluorescence or Forster resonance energy transfer (FRET) is widely accepted as being one of the most useful methods to observe biochemical events in vitro and in living cells. Generally, there are two forms of FRET sensors those based on a pair of genetically encoded fluorophores, usually employing fluorescent proteins from jellyfish or corals, or those based on small molecules that make use of small organic fluorophores. 

Energy transfer, as described by Forster [78], requires a long range dipole-dipole interaction between the donor and the acceptor fluorophore. This energy transfer is possible at distances between 2 and 10 nm. Contrary to what happens in collisional quenching, there is no need for physical contact between the two molecules. 

The rotation of the fluorophores is a factor that affects the energy transfer. Only maximal rotational freedom will permit tda estimation. There is no way to predict this factor. Therefore the dynamic averaged value of k2 is considered 2/3. This prediction induces a certain error in the calculation of distances (see Chap. 1). 

The term filterFRET here refers to intensity-based methods for calculating fluorescence resonance energy transfer (FRET) from sets of images of the preparation collected at different excitation and/or emission wavelength. The term is not intended to imply that interference filters are actually present in the setup very similar considerations apply when donor- and acceptor fluorophores are spectrally resolved by other means, such as monochromators or spectral detectors. 

Forster resonance energy transfer (FRET) is a form of quenching. For a fluorophore (donor) to be quenched by another molecule (acceptor), three criteria must be met ... 

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