I am recently working with a group of fluorophores. After I got optimal Em/Ex wavelengths, for each of them, I prepared a series of solutions with different concentration of fluorophore using PBS (containing 5% DMSO). Then, I measured fluorescence intensity of each solution using plate reader.
However, one fluorophore reached a fluorescence intensity plateau with a relative low concentration (~200 nM). When the plateau was reached, the intensity was just 200 AU.
Since I measured pure fluorophore solution. I thought the fluorescence intensity should keep increasing with the increase of fluorophore concentration. Can anyone have similar experience? or know anything related to this?
Thank you so much!
when the fluorophore concentration increases, , the scattering and reabsorption increase, too which leads to the plateau.
Generally, the fluorescence intensity increases in direct proportion to the fluorophore concentration when the fluorophore concentration is low. As the concentration increases, one of at least 3 things can happen: (1) the fluorophore solubility is exceeded, insoluble fluorophore aggregates or precipitates, and the fluorescence stops increasing, (2) the fluorophore self-quenches by forming complexes, (3) the fluorophore absorbs the exciting and possibly emitted light (the inner filter effect), reducing the amount of light detected. Saturation of the detector by too much light can also appear to cause a plateau.
with increasing fluorophore concentration, phenomena like reabsorption and/or innerfilter effects aare likely to come into play. This would lead to 'consumption' of truly fluorescence emitted photons in terms of being measured by the detector, the net result would appear like decrease of intensity eventually reaching a plateau. a detailed discussion on such issues could be helpful and for this a standard book directed to fluorescence spectroscopy may be consulted, e.g., the bok by Joe Lakowicz.
Please show spectra! ResearchGate makes it quite easy to attach a spectral plot to a posting...
It could be a consequence of
Fluorescence intensity due to a product must depend on its concentration, thus with increasing reactant conc. intensity incrases for the presence of higher amount fluorescence producing product at that reactant concentration. But, with the addition of higher and higher reactant the reaction attains equilibrium at certain concentrations according to La- Chatelier principle. After this intensity does not increases and becomes plateau
Hi Abhijit,
The explanation given by Adam, Bijan (and others as well) covers the scenario described by Alec - i.e. the fluorescence intensity as a function of a pure fluorophore.
Since there is no reaction involved, there are no products formed. The explanation you provided is valid for an enzyme catalyzed reaction with a fluorogenic substrate, in which case the "rate of increase in fluorescence intensity" reaches a plateau at saturating concentrations of reactant.
Hi Nitesh Sir,
Yes, you are true. I had probably not understand the actual question.
For a pure fluorophore at very high concentration, the excitation energy is transferred to nearby ground state molecule, i.e., fluorescence is decreased by 'static quenching' mechanism to reach plateau.
Adam B Shapiro explained the phenomenon you observed perfectly.
I would suggest you to run the following experiment: prepare a concentration series with the fluorophore you are interested in. Recdord the emission spectrum for each sample thus you can see any distortion of the baseline or aggregate formation, etc. Use in your experiment the concentration range where you find linearity between the concentration and fluorescence emission intensity.
Anyway, you can check the self-absorption phenomenon if you dilute the sample which reached plateau. If you get an increase in the fluorescence emission signal of the diluted sample, surely the concentration of the fluorophore was too high.
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