Stokes shift overlap

A consequence of the efficiency of vibrational relaxation is that the fluorescence band for a giv-bn-elec-tronic transition is displaced toward lower frequencies or longer wavelengths from the absorption band (the Stokes shift). Overlap occurs only for the resonance peak involving transitions between the lowest vibrational level of the ground state and the corresponding level of an excited state. In Figure 15-2, the wavelength of absorbed radiation that produces the resonance peak A, is labeled A[. 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

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. 

Since the same dye molecules can serve as both donors and acceptors and the transfer efficiency depends on the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, this efficiency also depends on the Stokes shift [53]. Involvement of these effects depends strongly on the properties of the dye. Fluoresceins and rhodamines exhibit high homo-FRET efficiency and self-quenching pyrene and perylene derivatives, high homo-FRET but little self-quenching and luminescent metal complexes may not exhibit homo-FRET at all because of their very strong Stokes shifts. 

A general way to predict the temperature dependence of the spectral overlap is not known. We found that /Py+/py+, Jqx-/ox. and also Jpy+/ox+ do not change significantly in the temperature range from 80 to 300 K (see Fig 1.26). The large difference between the absolute values of the overlap integrals /Py+/Py+, /0x+/Ox+ is due to a different Stokes shift (140 cm-1 for Ox+, 560 cm-1 for Py+). Other cases that show a temperature dependence of the spectral overlap also exist [3]. 

Representative absorption (excitation) and emission spectra of a fluoro-chrome are provided in Fig. 1. Some degree of overlap between the two spectra is typical, and often the excitation and emission spectra are mirror images of each other. The separation between the wavelengths at which excitation and emission maxima occur is referred to as the Stokes shift. 

Because of the Stokes shift for vibrationally relaxed systems (the rate of transfer < the rate of vibrational relaxation), transfer between like molecules is less efficient than that between unlike molecules when acceptor is at a lower energy level (exothermic transfer). No transfer is expected if the acceptor level is higher than the donor level. If (i) the acceptor transition is strong (Emaz —- 10,000), (ii) there is significant spectral overlap, and (iii) the donor emission yields lie within 0.1 — 1.0, then R0 values of 50-100 A are predicted. 

Figure 2a compares the time-resolved Stokes shift of the normal sequence and the abasic sequence. For ease of comparison, the data is shifted to overlap the sequences at early times. In the first nanosecond, the Stokes shifts from both sequences overlap almost perfectly. This results suggests that there is not a large scale collapse of the normal DNA structure at the abasic site. However after 1 ns, the abasic sequence has additional dynamics beyond those of the normal sequence. The fit of the abasic sequence has the same logarithmic component of the normal sequence fit, but with an additional exponential term for the fast rise in the Stokes shift after 1 ns S(t) = S0 + A0 logl0(t/t0) + 4,(l-exp(-f/r)), with an exponential time constant r of 25 ns. 

Exceptional fluorescence properties also characterize the ri.s-isomer 38e. Unsubstituted cis-l,2-di-9-anthrylethylene 38a and its monosubstituted derivatives such as 38b are nonfluorescent at room temperature. By contrast, cis-dianthrylethylene 38e does fluoresce with quantum yields of 0.0018, 0.0042, and 0.0064 in cyclohexane, dichloromethane, and acetonitrile, respectively. The emission is structureless (see Figure 18), and is associated with a solvent-independent Stokes shift of about 6000cm-1. As the molecular geometry of 38e is characterized by overlapping anthracene systems [80], the structureless emission may be attributable to an intramolecular excimer state. 

Figure 3.29 (a) Outline of the absorption, A fluorescence, F and phosphorescence, P spectra of a rigid polyatomic molecule. X = wavelength, vertical axis = absorbance (A) or emission intensity (F, P). (b) The Stokes shift of the absorption and fluorescence spectra is defined as the difference between their maxima. When this shift is small, there is a substantial spectral overlap between absorption and emission, (c) Jablonski diagram and outline of the absorption and fluorescence spectra of azulene, an exception to Kasha s rule. The energy gap between S0 and Sj is very small, that between Sj and S2 is very large... 

This fluorophore has an excitation maximum at 502 nm and an emission maximum at 510 nm. Tlie small Stokes shift of only 8 nm creates some difficulty in discrete excitation without contaminating the emission measurement with scattered or overlapping light. The extinction coefficient of the molecule in methanol is about 77,000 M-1cm-1 at 502 nm. 

Fig. 16.1. Typical geometry for common-path epi-scattering Raman measurements of biofluids, in vitro. The common path ensures that the excitation and collection volumes overlap (aside from slight changes in refractive index between the laser and Stokes-shifted wavelengths). A dichroic beamsplitter is required as drawn here, it is reflective at the Stokes-shifted wavelengths, but more commonly it is reflective at the laser wavelength...

Contrary to 6T, no indications for an additional luminescence from deep traps are found in the 4T spectra. The reason for this difference between 4T and 6T may be that in the case of 4T one type of structure is energetically more strongly favored than in the case of 6T, possibly due to the reduced molecular size of 4T. In agreement with the above finding, the 0-0 transition of 4T in photoluminescence (21,025 cm-1) is observed to be in resonance with the lowest line observed in absorption (21,035 cm-1). Since the lines have a considerable overlap, the Stokes shift is below 10 cm-1. 

There are experimental evidence for the assignment of Sj to the ttct state for both HFB and PFB. Figure 15-33(a) presents the fluorescence excitation and dispersed fluorescence spectra of HFB in supersonic free jet [74], The fluorescence excitation spectra very closely mimic the vapor-phase irir S0 absorption spectra of the compound. It is evident that there is no spectral overlap between the fluorescence and the tht <- S0 absorption spectra of HFB. The energy difference between the absorption and emission maxima is greater than 11 000 cm-1. Moreover, the full width at half maximum (FWHM) of the absorption is about 3000 cm-1, whereas that of the dispersed emission is about 5500 cm-1. For fluorinated benzenes with four or less F atoms, the absorption and emission bands overlap with the Stokes shift of about 4000 cm-1, and the FWHM of both bands is about 3000 cm-1. The FWHM absorption bandwidth of 3000 cm-1 is characteristic of tht (Lft) S0... 

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