Excitation spectrum, fluorescence

Since the sensitivity of fluorescence is higher than absorption, the fluorescence excitation spectrum of a pure molecule can be obtained at low concentrations compared to the absorption spectrum. 

Emission and excitation spectra were recorded at A.ex = 300 nm and A.em = 420 nm, respectively. 

The excitation spectrum is technically perturbed by two problems the light intensity of the excitation lamp, which varies with the wavelength, and the intensity upon detection, which is also wavelength-dependent. Using rhodamine B dissolved in glycerol as a reference could perform corrections. Radiation from rhodamine is proportional to the excitation intensity independently of the excitation wavelengths. Therefore, excitation of rhodamine will yield a fluorescence excitation spectrum that characterizes the spectrum of the excitation lamp. In order to obtain the real fluorescence excitation spectrum of the studied fluorophore, the recorded excitation spectrum will be divided by the excitation spectrum obtained from the rhodamine. This procedure is done automatically within the fluorometer. 

In general, when one wants to find out if structural modifications have occurred within a protein, circular dichroisni experiments are performed. This will help to find out whether the global structure of the protein or / and its local stmcture have been altered or not. However, one can record also the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from a state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

For example, we studied the interaction between the fluorescent probe calcofluor and a 1-acid glycoprotein at two concentrations of calcofluor. Also, we recorded the fluorescence excitation spectrum of the protein so that to find out whether binding of calcofluor to the protein modifies its structure or not. 

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Figure Bl.1.4. Two-photon fluorescence excitation spectrum of naphthalene. Reprinted from [35], Courtesy, Tata McGraw-Hill Publishing Company Ltd, 7 West Patel Nagar, New Dehli, 110008, India.

Figure B2.3.15. Laser fluorescence excitation spectrum of the A S -X ff (1,3) band for the OH product, in the V = 3 vibrational level, from tire H + NO2 reaction [44]- (By pemrission from AIP.)...

Figure 9.29 Two-photon fluorescence excitation spectrum of 1,4-difluorobenzene. The upper and lower traces are obtained with plane and circularly polarized radiation, respectively, but the differences are not considered here. (Reproduced, with permission, Ifom Robey, M. J. and Schlag, E. W., Chem. Phys., 30, 9, 1978)...

Nevertheless, 1,4-difluorobenzene has a rich two-photon fluorescence excitation spectrum, shown in Figure 9.29. The position of the forbidden Og (labelled 0-0) band is shown. All the vibronic transitions observed in the band system are induced by non-totally symmetric vibrations, rather like the one-photon case of benzene discussed in Section 7.3.4.2(b). The two-photon transition moment may become non-zero when certain vibrations are excited. 

Electronic transitions in molecules in supersonic jets may be investigated by intersecting the jet with a tunable dye laser in the region of molecular flow and observing the total fluorescence intensity. As the laser is tuned across the absorption band system a fluorescence excitation spectrum results which strongly resembles the absorption spectrum. The spectrum... 

Figure 9.48 Part of the fluorescence excitation spectrum of 1,2,4,5-tetrafluorobenzene in a supersonic jet. (Reproduced, with permission, from Okuyama, K., Kakinuma, T, Fujii, M., Mikami, N. and Ito, M., J. Phys. Chem., 90, 3948, f986)...

Fig. 36. Laser fluorescence excitation spectrum of 7-azoindole dimer (6.1).

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Fig. 4.1.16 Luminescence spectrum of aequorin triggered by Ca2+ (solid line /.max 465 nm), and the fluorescence spectra of Aequorea GFP excitation (dashed line A.max 400 nm and 477 nm) and emission (dash-dot line 7max 509 nm). The dotted line is the fluorescence excitation spectrum of GFP in the light organs, showing that 480 nm excitation peak is almost missing — an evidence showing that GFP in light organs exists in an aggregated form having a very low E value at 480 nm.

The fluorescence excitation spectrum of a Q-CdS sample, with several maxima in the absorption spectrum, also has a number of peaks. However, the maxima in the two spectra do not always occur at the same wavelengths This effect is not surprising, as excitation at different wavelengths leads to the excitation of particles of different sizes which do not have the me fluorescence intensity at the wavelength where the fluorescence is recorded. 

To determine the fluorescence excitation spectrum, one selects a wavelength on the emission monochromator (generally the fluorescence A 1 and... 

It is possible, however, that the electrochromic response of some styrylpyridi-nium probes, for example, RH421 (see Fig. 2), is enhanced by a reorientation of the dye molecule as a whole within the membrane. There is a steep gradient in polarity on going from the aqueous environment across the lipid headgroup region and into the hydrocarbon interior of a lipid membrane. Therefore, any small reorientation of a probe within the membrane is likely to lead to a change in its local polarity and hence a solvatochromic shift of its fluorescence excitation spectrum. Such a... 

In methanol/DMSO solvent mixtures the fluorescence spectrum of TIN (A.max = 400 nm) displays a normal Stokes shift indicating that this emission arises from a non proton-transferred, excited state of TIN. The fluorescence excitation spectrum for this emission coincides with the absorption spectrum of the resolved non-planar species suggesting that this conformer is the ground-state precursor responsible for the observed emission. As the amount of DMSO in the mixture increases the fluorescence maximum undergoes a bathochromic shift from 415 nm in pure methanol to 440 nm in pure DMSO. 

The values of ftot for various benzotriazole compounds in a range of solvents are listed in Table II. Values of the fluorescence quantum yield for TIN and TINS, corrected for the absorbance by their non-fluorescent, planar conformers at the excitation wavelength, are listed in Table III. In all the benzotriazole solutions examined, maximum fluorescence emission was observed at about 400 nm indicating that this emission originates from the non proton-transferred species. This was confirmed by examination of the fluorescence excitation spectrum which corresponds to the absorption spectrum of the non-planar form of the molecule. 

The fluorescence emission spectrum of TIN in PS and PMMA films is dominated by a blue emission with a maximum at approximately 400 nm. The fluorescence excitation spectrum, monitoring this emission, corresponds to the absorption band of TIN(non-planar) obtained from the PCOMP analysis indicating that it is this form that leads to the observed emission. 

Now we will assume in addition that the total measured fluorescence is proportional to the above volume average. This can be accomplished experimentally by suspending the fluorescent particle in an integrating enclosure and monitoring the fluorescence with an optical fiber which is pushed through a small hole in the side of the enclosure. Our interest in what follows is to use Eq. (8.4) to simulate a fluorescence excitation spectrum. 

Figure 8.1S. Spectra of a glycerol coated with a fraction of a monolayer of dil(S). The upper curve is the fluorescence excitation (/,) spectrum, and the lower curve is the 90° elastic scattering (/,) spectrum.

Fig. 6 Bottom-up (i) fluorescence excitation spectrum of the 1 1 diastereomeric complexes between (5)-2-naphthyl-l-ethanol (F ) and 2-butanol (M /M ) (ii) hole-burning spectrum obtained with the probe tuned on the transition located at - 136 cm ([F M ] complex) (iii) (c) hole-burning spectrum obtained with the probe tuned on the transition located at — 69 cm ([F -Ms] complex) (iv) hole-burning spectrum obtained with the probe tuned on the transition located at — 73 cm ([F -M/ ] complex). The probed band is denoted by A. The bands due to the bare chromophore are denoted by 2-NEtOH (reproduced by permission of the American Chemical Society).

In pure crystals, singlet excitons can be created by mutual annihilation of triplet excitons. The intensity of the singlet exciton fluorescence depends quadratically on the triplet exciton concentration and is therefore proportional to the square of the singlet-triplet extinction coefficient. It is interesting to compare such a delayed fluorescence excitation spectrum, observed by Avakian et cd. 52) on naphthalene, with a corresponding phosphorescence excitation spectrum (Fig. 22). 

Fig. 9. Fluorescence excitation spectrum (solid line) and fluorescence emission spectrum (dotted line) of PplX in methanol (kindly provided by B. Ebert, Physikalisch-Technische Bundes-anstalt, Berlin)...

Physical Properties Density average and standard deviation Size average and standard deviation Fluorescence Fluorescence excitation spectrum Scattered light spectrum Absorption spectrum (from microwave to UV) Raman spectrum Electrical conductivity, impedance Acoustic properties... 

The term 2PA spectrum will be used here and in the rest of the chapter to indicate the variation of the two-photon cross section as a function of wavelength, even when the data were obtained by monitoring the intensity of the fluorescence emission induced by two-photon absorption (that is when, strictly speaking, the two-photon induced fluorescence excitation spectrum was obtained). Also, unless otherwise specified, the spectra are reported as a function of the wavelength of the excitation beam and are degenerate 2PA spectra. 

To measure a fluorescence excitation spectrum, a band of fluorescence from a solute in C was selected by the monochromator M2 and was received by the photomultiplier After amplification the outputs of Pi and P2 were passed to the ratio recorder, R. The frequency drum on Mi was motor-driven and as the frequency was varied the slits were adjusted so as to maintain the output of P2 approximately constant. If the contents of F were so chosen as to make the output of P2 proportional to the quantum output of Mj at all frequencies, then since Pi was proportional to ht (see eq. 2) and P2 was proportional to I0, the recorded ratio was proportional to t, i.e., the true excitation spectrum was recorded. 

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