Fig 1.
Comparison between the conventional fluorometric system and the nano-cuvette system.
(a) Fluorescence measurement of solutions with low and high fluorophore concentrations in a microplate reader. Compared with solutions of low concentration in which all the fluorophores are excited (left), at high concentration, only a small portion of fluorophores near-surface are excited owing to the rapid absorption of the excitation light. (b) Schematic representation of the nano-cuvette system. The excitation beam illuminates the chamber in total internal reflection mode which generates an evanescence field with a penetration depth of ~100 nm. With this depth, the emission from all the fluorophores can be collected by the objective lends as no absorption of excitation beam occurs.
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.
Fig 3.
Degree of quenching against concentration.
All of the curves were fitted with the modified Stern-Volmer equation for self-quenching: . The value of ν was 2.6, for Sulforhodamine B, 3.0 for FTIC and 0.04 for calcein.