Correction fluorescence intensity

Inner filter effects due to the presence of other substances When the solution contains other chromophores that absorb light in the same wavelength range as the fluorescent compound under study, the chromophores act as filters at the excitation wavelength and the fluorescence intensity must be multiplied by a correction factor. If the chromophores do not interact with the fluorescent compound, the correction factor is simply the fraction of light absorbed by the compound at the chosen excitation wavelength, so that the corrected fluorescent intensity is given by ... 

One can conclude that Equation (7.15) is very useful in correcting fluorescence intensities recorded at high ODs. Also, it is important to indicate that the curvature observed, at high ODs, in the experimental plot is not the result of fluorescein aggregation, since ODs increase linearly with fluorescein concentrations (Figure 9.3). 

Measure the fluorescence intensity at the peak, correct it for dilution, then for the inner filter effect, and plot the corrected fluorescence intensity as a function of added DNA concentration. Use the data to determine the number of binding sites and the association constant of the complex. 

Calibration graph (d) may be used to correct for the small fluorescence intensity due to morphine in NaOH this is not negligible when the morphine concentration is high and the codeine concentration is low. 

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.

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. 

Emission ratio imaging is extremely popular due to its simplicity and speed. In essence, cells expressing donors and acceptors are illuminated at the donor wavelength and fluorescence intensity data are collected both at donor (D) and at acceptor (S) channels. Collected data may be either images, or, in case high acquisition speed is crucial and spatial information is not required, dualchannel photometer readings (see Textbox 1). S and D are not overlap-corrected and FRET is simply expressed as the ratio of intensities1 as ratio = S/D. 

Fig. 7.7. Effects of Poisson photon noise on calculated SE and FRET values. (A) Statistical distribution of number of incoming photons for the mean fluorescence intensities of 5,10, 20, 50, and 100 photons/pixel, respectively. For n = 100 (rightmost curve), the SD is 10 thus the relative coefficient of variation (RCV this is SD/mean) is 10 %. In this case, 95% of observations are between 80 and 120. For example, n — 10 the RCY has increased to 33%. (B) To visualize the spread in s.e. caused by the Poisson distribution of pixel intensities that averaged 100 photons for each A, D, and S (right-most curve), s.e. was calculated repeatedly using a Monte Carlo simulation approach. Realistic correction factors were used (a = 0.0023,/ = 0.59, y = 0.15, <5 = 0.0015) that determine 25% FRET efficiency. Note that spread in s.e. based on a population of pixels with RCY = 10 % amounts to RCV = 60 % for these particular settings Other curves for photon counts decreasing as in (A), the uncertainty further grows and an increasing fraction of calculated s.e. values are actually below zero. (C) Spread in Ed values for photon counts as in (A). Note that whereas the value of the mean remains the same, the spread (RCV) increases to several hundred percent. (D) Spread depends not only on photon counts but also on values of the correction...

The variations in fluorescence intensity as a function of the excitation wavelength XE for a fixed observation wavelength represents the excitation spectrum. According to Eq. (3.20), these variations reflect the evolution of the product /0(FE)A(AE). If we can compensate for the wavelength dependence of the incident light (see Chapter 6), the sole term to be taken into consideration is A(AE), which represents the absorption spectrum. The corrected excitation spectrum is thus identical in... 

When the incident light is horizontally polarized, the horizontal Ox axis is an axis of symmetry for the fluorescence intensity Iy = Iz. The fluorescence observed in the direction of this axis (i.e. at 90° in a horizontal plane) should thus be unpolarized (Figure 5.3). This configuration is of practical interest in checking the possible residual polarization due to imperfect optical tuning. When a monochromator is used for observation, the polarization observed is due to the dependence of its transmission efficiency on the polarization of light. Then, measurement of the polarization with a horizontally polarized incident beam permits correction to get the true emission anisotropy (see Section 6.1.6). 

For analytical applications, when a linear relationship between fluorescence intensity and concentration is desirable, a correction curve must be built up under the same conditions as those that will be used for the actual experiment. 

The spectral overlap is an important quantity in radiationless energy transfer and migration, as we have seen in Eq. (32). It is equal to the integral of the corrected and normalized fluorescence intensity if (v) of the donor multiplied... 

Notice that the fluorescence intensity is directly proportional to the optical density only over a narrow range. Use of the above equation corrects for this apparent loss in fluorescence intensity. 

The relation between fluorescence intensity and structure was studied by Goldzieher et al. [113]. For this work, 0.2 mL of an ethanolic steroid solution was added to 1 mL of 90% (v/v) sulfuric acid, the mixture heated at 80°C for 10 minutes, and finally diluted with 4.0 mL of 65% (v/v) sulfuric acid. The resultant solution contained 25-250 pg/ml, and was measured at an excitation wavelength of 436 nm. No correction was made for self-absorption of the solutions. 

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