Fluorescence corrected spectra

Fig. 4.2.2 Left panel-. Uncorrected Ca2+-triggered bioluminescence spectrum of W92F obelin derived from O. longissima. Right panel Corrected bioluminescence spectrum of the same obelin (dotted line), and the fluorescence emission spectrum of the spent solution after luminescence (solid line). From Deng et al., 2001, with permission of the Federation of the European Biochemical Societies.

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

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 quantum yield of a compound may be determined by comparing the area under its fluorescence spectrum with the area under the fluorescence spectrum of a reference compound whose fluorescence quantum yield is known. The spectra of both compounds must be determined under the same conditions in very dilute solution using a spectrometer incorporating a corrected spectrum capability, in order to overcome any variation in detector sensitivity with wavelength. 

A fluorescence emission spectrum is generally measured by setting the excitation monochromator, Mi, to the chosen wavelength and scanning the second monochromator, M2, with constant slit width. The fluorescent screen monitor, F-P2, now serves to correct for variations in the intensity of the exciting light caused by fluctuations in lamp output. The emission spectrum so recorded has to be corrected for the spectral sensitivity of the apparatus to give the true emission spectrum. 

Figure 23-13 (A) Corrected emission and excitation spectra of riboflavin tetrabutyrate in w-heptane. Concentration, about 0.4 mg I-1. Curve 1 excitation spectrum emission at 525 nm. Curve 2 emission spectrum excitation at 345 nm. FromKotaki and Yagi.128 (B) Indole in cyclohexane, T = 196 K. 1, Fluorescence excitation spectrum 2, fluorescence spectrum and 3, phosphorescence spectrum. From Konev.125...

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. Corrections can be performed using rhodamine B, dissolved in glycerol, as reference. In fact, 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 excitation lamp spectrum. 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 rhodamine. This procedure is done automatically within the fluorometer. 

In general, when one wants to determine if global and/or local structural modifications have occurred within a protein, circular dichroism experiments are performed. Also, one can record the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from one state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

Delayed fluorescence from a very-short-lived upper excited singlet state populated by hetero-TTA has been observed for the first time using the system A = anthracene and X = xanthone (Nickel and Roden, 1982). An energy-level diagram for this system is shown in Figure 5.32, and the corrected spectrum of the delayed fluorescence of anthracene and xanthone in trichlorotrifluoroe-thane is depicted in Figure 5.33. The band at 36,000-40,000 cm has been assigned to the delayed fluorescence of anthracene produced by Tf +... 

Figure 2 Corrected fluorescence emission spectrum of Sq4 in various pentanols (cone. 3 X lO- M)-...

True spectrofluorometcrs allow production of a fluorescence excitation spcclruni or a fluorescence emission spectrum. Figure 1.5-9a shows an excitation spectrum for anthracene in which the fluorescence emission was measured at a fixed wavelength while the excitation wavelength was. scanned. With suitable corrections for variations in source output intensity and detector response as a function of wavelengtli, an absolute excitation spectrum is obtained that closely resembles an absorption spectrum. 

The efficiency of photon detection of the emission monochromator and the photomultiplier is not 100% at all wavelengths. Therefore, the recorded emission spectrum can be distorted at many wavelengths. Corrections can be done by comparing the recorded spectrum of the sample to the recorded spectrum of a standard whose corrected fluorescence emission spectrum is known. Also one can use standard lamps whose real outputs is known. Comparison of the lamp output obtained with the laboratory material with the real one allows calculating the sensitivity of the fluorometer at every wavelength. 

Raman spectra of S2 in its triplet ground state have been recorded both in sulfur vapor and after matrix isolation using various noble gases. The stretching mode was observed at 715 cm in the gas phase [46], and at 716 cm in an argon matrix [71]. From UV absorption and fluorescence spectra of sulfur vapor the harmonic fundamental mode of the S2 ground state was derived as t e = 726 cm . The value corrected for anharmonicity is 720 cm [26, 27]. Earlier reports on the infrared absorption spectrum of 2 in matrix isolated sulfur vapor [72] are in error the observed bands at 660, 668 and 680 cm are due to S4 [17] and other species [73]. 

Photophysical Processes in Dimethyl 4,4 -Biphenyldicarboxy-late (4,4I-BPDC). The ultraviolet absorption spectrum of dimethyl 4,4 -biphenyldicarboxyl ate was examined in both HFIP and 95% ethanol. In each case two distinct absorption maxima were recorded, an intense absorption near 200 nm and a slightly less intense absorption near 280 nm. The corrected fluorescence excitation and emission spectra of 4,4 -BPDC in HFIP at 298°K shows a single broad excitation band centered at 280 nm with a corresponding broad structureless emission band centered at 340 nm. At 77°K, the uncorrected phosphorescence spectra shows a single broad structureless excitation band centered at 298 nm, and a structured emission band having maxima at 472 and 505 nm with a lifetime, t, equal to 1.2 seconds. 

Photophysical Processes in Pol,y(ethy1eneterephthalate-co-4,4 -biphenyldicarboxyl ate) (PET-co-4,4 -BPDC). The absorption and luminescence properties of PET are summarized above. At room temperature the absorption spectrum of PET-co-4,4 -BPDC copolymers, with concentrations of 4,4 -BPDC ranging from 0.5 -5.0 mole percent, showed UV absorption spectra similar to that of PET in HFIP. The corrected fluorescence spectra of the copolymers in HFIP exhibited excitation maxima at 255 and 290 nm. The emission spectrum displayed emission from the terephthalate portion of the polymer, when excited by 255 nm radiation, and emission from the 4,4 -biphenyldicarboxylate portion of the polymer when excited with 290 nm radiation. 

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