Fluorescence resonance energy spectral overlap

Takakusa H, Kikuchi K, Urano Y, Kojima H, Nagano T (2003) A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral. Chemistry 9 1479-1485... 

Fig. 12 Schematic representation of Fluorescence Resonance Energy Transfer (FRET). The excitation of the donor occurs at wavelength Xi. The good spectral overlap of donor emission and acceptor excitation allows the transfer of the excitation from donor to acceptor. The acceptor emits at the longer wavelength X2.

Fluorescence Resonance Energy Transfer (FRET), Fig. 1 (a) Jablonski diagram illustrating FRET and related processes, including excitation of the donor, radiative (solid line) and non-radiative (dashed lines) relaxation on the donor and acceptor, vibrational relaxation (short curved arrows), and transitions associated with FRET (dotted lines). Processes that determine the FRET efficiency are indicated in bold, (b) Illustration of spectral overlap between Cy3 (donor) emission and Cy5 (acceptor) absorption, (c) Definition of the angles used to calculate... 

Figure 3. Fluorescence Resonance Energy Transfer in pH sensing. The energy transfer is dependent upon the distance between donor and acceptor molecules, the extent of spectral overlap between donor emission and acceptor absorbance, the quantum yield of the donor and extinction coefficient of the acceptor.

Our previous approaches to detect endogenous complexes of dynamin and auxilin using co-immunoprecipitation approaches were unsuccessful, so we turned to fluorescence lifetime imaging microscopy (FLIM). While fluorescence microscopy provides two- or three-dimensional information about fiuorophore concentration, FLIM can reveal spatial differences in fluorophore population lifetimes that are independent of concentration. Besides being useful in fiuorophore identification, which transcends issues of spectral overlap, FLIM inherently observes lifetime truncations on a pixel by pixel basis that are induced by fluorescence resonance energy... 

Figure 10. Fluorescence resonant energy transfer (FRET) of single fluorophores pairs, (a) If two molecules with different absorption and emission properties are in close proximity (<10 mn), the fluorophore with the higher emission energy (bluer wavelength, donor ) can transfer its excitation energy to a second fluorophore ( acceptor ), (b) This radiationless energy-transfer process depends on the spectral overlap between the donor and acceptor emission and absorption, as well as their spatial separation and the alignment of their dipole...

TEOS nanoparticles doped with three types of dye were prepared for fluorescence-resonance energy transfer (FRET) by means of imposition [55]. The dyes fluorescein, 5-carboxyrhodamine 6G (R6G) and 6-carboxy-X-rhodamine (ROX) were used to prepare these TEOS nanoparticles because their spectral features effectively overlap. In the triple-dye-doped TEOS nanoparticles, fluorescein was used as a common donor for R6G and ROX, whereas R6G acted both as an acceptor for fluorescein isothiocyanate (FITG) and as a donor for ROX. To prepare the nanoparticles, the three types of amine-reactive dye molecule were first covalently linked to APS, 5-(and-6)-carboxyfluorescein succinimidyl ester, 5-carboxyrhodamine 6G succinimidyl ester and 6-carboxy-X-rhodamine succinimidyl ester. 

The Na+ sensor M-9 has a structure analogous to that of compound E-4, but instead of two identical pyrene fluorophores, it contains two different fluorophores with a pyrene group and an anthroyloxy group. Resonance energy transfer (see Chapter 9) from the former to the latter is then possible because of the spectral overlap between the fluorescence spectrum of the pyrene moiety and the absorption spectrum of the anthroyloxy moiety. Upon addition of Na+ to a solution of M-9 in a mixture of MeOH and THF (15 1 v/v), the fluorescence of the anthroyloxy group increases significantly compared with that of the pyrene group, which permits a ratiometric measurement. 

From the above equations, it can be seen that in dipole resonance transfer, the rate of transfer is dependent on the fluorescence intensity and on the fluorescence lifetime of the donor, as well as on the spectral overlap between the fluorescence of the donor and the absorbance of the acceptor. Two very important features of this transfer mechanism are the sixth-power dependence on the separation of the two molecules as well as the fact that it is possible to transmit energy by resonance transfer over distances of up to 50 A, a distance corresponding to several molecular diameters. ... 

Here pis the quantum efficiency of the sensitizer (ti = Tp/xp = l/3forpentacene)in the O4 site of p-terphenyl at 4 K, n is the index of refraction (n = 1.7 for the p-terphenyl crystal), and Na is the Avogadro s number. The integral in (H9) is calculated from the normalized fluorescence spectrum/(v) and the decadic molar extinction coefficient e(v) of pentacene at O4 site. The critical interaction distance is the sensitizer-activator separation for which the transfer rate is equal to the intrinsic decay time. Although derived for low temperatures. Equation H9 is also vaUd for arbitrary temperatures. In fact, the temperature dependence of the resonant energy transfer rate is contained in the spectral overlap integral. 

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