Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field

The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon-called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 μM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.