Fluorophores feature

Other naturally occurring fluorescent proteins have been found in aquatic organisms including reef corals and sea anemones. The red fluorescent protein DsRed was discovered in the Anthozoan genus Discosoma. The DsRed fluorescent protein fluorophore features an imidazoline ring system similar to the GFP fluorophore but... 

A useful division of the contributing mechanistic features of any chemiluminescence process involving excitation of a "spectator" fluorophore is given in Equation 2. To optimize the chemical efficiency > we have also undertaken a systematic investigation of... 

Another important feature of fluorophores is the amount of vibrational energy lost in the excited state. The difference between emission and excitation maxima gives a readout in this respect and is referred to as the Stokes shift. In many sensors, a small Stokes shift is unfavorable for FRET ratio measurements due to overlap of emission spectra. 

An important feature that influences the performance of the probe to a large extent is the selection of the FRET pair. Generally, fluorophores with high excitation wavelengths are preferred over blue fluorophores in single photon microscopy for applications in tissue since there is no autofluorescence in this region and the ratio... 

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

One of the most characteristic features of FRET is its sensitive dependency on the fluorophore distance. This is advantageously used to evaluate structures and conformational changes of peptides, glycopeptides, and proteins among other molecules [164-166], The conformational change of the lipopeptide antibiotic daptomycin from an inactive linear form to a biological active cyclic form... 

Stepping outside of the subject of biochemical protein dynamics there is also a healthy literature on the use of sensing using fluorophores. Spectral and lifetime characteristics of fluorophores are dependent on their environment, for example, pH, O2, and Ca2+, these features are a useful tool, particularly in the study of the basic biology of the cell (see for instance Chapter 4). 

In contrast to brightfield microscopy, which uses specimen features such as light absorption, fluorescence microscopy is based on the phenomenon in which absorption of light by fluorescent molecules called fluorescent dyes or fluorophores (known also as fluorochromes) is followed by the emission of light at longer... 

Another feature is that the absence of spectral change precludes ratiometric measurements. However, dual-wavelength Cl sensors have been constructed. For instance, in compound A-6 (Figure 10.30), 6-methoxyquinolinium (MQ) as the Cl -sensitive fluorophore (blue fluorescence) is linked to 6-aminoquinolinium (AQ) as the Cl--insensitive fluorophore (green fluorescence), the spacer being either rigid or flexible. 

Some features of the spectra obtained with conventional equipment are illustrated with unsaturated hydrocarbons as fluorophores that have been adsorbed from gas or liquid phase on highly porous metal oxide powders as scattering substrates. 

Heterogeneous fluorescence immunoassays have many different assay formats, but all possess one unifying feature the free fluorophore remaining in solution after binding of some of the fluorophore to antibody must be removed before quantification can be achieved. Again, both negative and positive reading assay formats have been employed. 

Another feature of the simplest model that needs modification is the assumption of a fixed dipole amplitude. Because of the efficient capture of nonpropagating near fields by a surface, a fixed-amplitude dipole emits more power, the closer it moves to a surface. However, in steady-state fluorescence, the emitted power can only be as large as the (constant) absorbed power (or less, if the intrinsic quantum yield of the isolated fluorophore is less than 100%). Therefore, the fluorophore must be modeled as a constant -power (and variable-amplitude) dipole. Many of the earlier theoretical references listed above deal only with constant-amplitude dipoles, so their results must be considered to be an approximation. 

The two above features which modify the simplest theory extend the range of distances z between the fluorophore and the surface over which the results remain valid, from a minimum of several hundred nanometers without the modifications to less than ten nanometers with them. Those two features are incorporated into the results displayed here. Other refinements, not included here, involve consideration of energy transfer to electron-hole pairs (for metals only at z < 10 nm) and nonhomogeneous atomic field effects (z<0.25 nm). We first assume that the intrinsic quantum yield is 100% then we will modify that assumption. 

The polarization properties of the evanescent wave(93) can be used to excite selected orientations of fluorophores, for example, fluorescent-labeled phosphatidylethanolamine embedded in lecithin monolayers on hydrophobic glass. When interpreted according to an approximate theory, the total fluorescence gathered by a high-aperture objective for different evanescent polarizations gives a measure of the probe s orientational order. The polarization properties of the emission field itself, expressed in a properly normalized theory,(94) can also be used to determine features of the orientational distribution of fluorophores near a surface. 

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