I would like to know, is there any literature on the effect of fluorophore concentration on the emission spectra? Particularly, I wants to know the Alexa fluor 488/GFP/fluorescein dilution in PBS.
At low concentrations (sub-micromolar to micromolar, depending on the compound), the emission spectrum is likely to be independent of the fluorophore concentration, except that the intensity increases with concentration. However, once the concentration increases so much that the absorbance of the compound becomes significant, the absorption of light will affect the emission spectrum, typically by preferentially absorbing the shorter wavelengths. In addition, the fluorophore can show self-quenching due to interactions between molecules.