Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field
Fig 2
Normalized fluorescence signal per fluorophore at different concentrations recorded with conventional fluorometer and TIR microscope.
Fluorescence signals from the conventional fluorometer are significantly underestimated by approximately two to three orders of magnitude (black dots). In TIR, 1000-fold self-quenching is observed at 50 mM sulforhodamine B solution (a) and 100 mM fluorescein isothiocyanate (FITC) solution (b). Calcein shows only 100-fold quenching at 50 mM concentration (c). Raw data can be found in S1 Table in S1 File.