In Stern-Volmer equation, are F and F0 the intensities taken at the same wavelength from emission spectra or the maximal intensities of the each curve? For example, how to calculate quenching rate or binding constants if fluorescence quenching is accompanied with red or blue shift?
The Stern Volmer plot is the plot is that of Io/I vs Concentration as said . But it can be also expressed as (Io-I)/I=K[Q]. This is a particular case of the plot of (Io-I)/(I-I∞)=K[Q] where I∞ is the remaining intensity for an infinite concentration of quencher [Q]. It can be the blank fluorescence, if any, or the fluorescence of the quenched fluorophore, if any. This last relation is quite general since (Io-I) is proportional to [FQ] and (I-I∞) is proportional to [F] as long as the signal I, fluorescence, absorbance, scattering is proportional to concentration. Interestingly you can have a straight line in two cases : if you have a static quenching mechanism or a dynamic quenching mechanism. In your case a static quenching is the more probable. You will not get a straight line if both mechanisms act together. Indeed there is non more a linearity between concentrations and signals.
The y-axis is the ratio of the unquenched fluorescence intensity divided by the quenched fluorescence intensity. The x-axis is the quencher concentration. The y-intercept is 1. The slope is the Stern-Volmer constant.
Dear, @Mohamed Khedawy , I am kindly grateful for detailed answer. I understand what is I and I0. But except quenching I have a slight (about 15 nm) red shift in fluorescence emission spectra. In this case I'm asking what must be taken for I and I0 : y-coordinates of the peaks(which are shifted with respect to each other) of y-coordinates at the same wavelength?
y coordinates of the peak should be taken. Or take area under the curve as I.
Hi Andrii Lesiuk ,
Well, that is an intriguing question because red or blue shifts in the emission spectra is usually interpreted as changes in the environment of the fluorescent probe. Such changes affect the electronic distribution and consequently modify the energy gaps, which reflects in the change of the wavelength emitted.
In that case, I agree with Nilimesh Das , I would take the maximal intensities of each curve, because they are referent to the specific electronic transition that you are tracking, the shift is a secondary effect... Another point is that if you take F0 and F at the same wavelength you will be overestimating the quenching. I think this work can help you: Vivian, James T., and Patrik R. Callis. "Mechanisms of tryptophan fluorescence shifts in proteins." Biophysical journal 80.5 (2001): 2093-2109.
And last but not the least, are you working with a low concentration of quenchers? Did you correct the inner filter effect? I have published results of fluorescence experiments in which a saw shift in the emission spectra, but when I corrected the inner filter, the shift disappeared (Povinelli, Ana Paula Ribeiro, et al. "Details of the cooperative binding of piperlongumine with rat serum albumin obtained by spectroscopic and computational analyses." Scientific reports 9.1 (2019): 1-11.). In that case, the quencher absorbed the light according to its own absorbance spectra causing an “optical ilusion”. In this work we showed the possibility of building Stern-Volmer plot based on the area under the spectra.
I agree with Nilimesh Das, You can consider the peak with max intensities in case of red or blue shift
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