Fluorophores resonance interactions

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 resonance energy transfer (FRET) has also been used very often to design optical sensors. In this case, the sensitive layer contains the fluorophore and an analyte-sensitive dye, the absorption band of which overlaps significantly with the emission of the former. Reversible interaction of the absorber with the analyte species (e.g. the sample acidity, chloride, cations, anions,...) leads to a variation of the absorption band so that the efficiency of energy transfer from the fluorophore changes36 In this way, both emission intensity- and lifetime-based sensors may be fabricated. 

More sophisticated designs involved semiconductor quantum dots with fluorescent protein receptors immobilized on the surface [146], The binding site of the protein receptor is occupied with an efficient fluorophore. On excitation a series of FRET (Forster resonant energy transfer) processes takes place excitation energy is transferred from the core of the quantum dot to the fluorescent protein and subsequently to the fluorophore. On substrate binding only one FRET step takes place and luminescence of the receptor is observed [146], In the simplest sensor architecture the protein contains bound quencher. Upon interaction with analyte the quencher is liberated and luminescence of the quantum dot is observed (Figure 16.25c). 

While the capture on DNA chips of fluorophore-labelled targets, and the extension of arrayed primers with fluorophore-labelled nucleotides has been widely used for some time, it is only more recently that assay formats have developed that utilize immobilized nucleic acids already modified with fluorophores. Fundamental analyses of surface monolayer structures and chemistries can be readily performed by immobilizing such modified oligonucleotides into SAM structures [105,106], but it is those interactions that can be monitored using fluorescence quenching or fluorescence resonance energy transfer (FRET) that have gained the most attention. 

Several other studies (150-153) reported that metal surfaces were able to either enhance or suppress the radiative decay rates of fluorophores. Furthermore, an increase in the extent of resonance energy transfer was also observed. These effects might be due to the interactions of excited-state fluorophores with SPs, which in turn produce constructive effects on the fluorophore. The effects of metallic surfaces include fluorophore quenching at short distances ( 0-5 nm), spatial variation of the incident light field (-0-15 nm), and changes in the radiative decay rates (-0-20 nm) (64). The term of metal-enhanced fluorescence could be referred to the appplication of fluorophore and metal interactions in biomedical diagnosis (64). 

He L, Olson DP, Wu X, Karpova TS, McNally JG, Lipsky PE. A flow c)4ometric method to detect protein-protein interaction in living cells by directly visualizing donor fluorophore quenching during CFP-YFP Fluorescence Resonance Energy Transfer (FRET). Cytometry 2003 55A 71-85. 

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