Parametric investigations on the saturation intensity of Coumarin 102 for stimulated emission depletion application

Stimulated emission depletion (STED) microscopy performed using continuous-wave (CW) lasers has been investigated and developed by Willig et al. (Nature Methods, 2007, 4(11):915) for nearly a decade. Kuang et al. (Review of Scientific Instruments, 2010, 81:053709) developed the CW STED microscopy technique with 405 nm excitation and 532 nm depletion beams. In their research, Coumarin 102 dye was adopted and was found to be depletable. In this study, a parametric investigation of the depletion of Coumarin 102 dye is carried out experimentally. The influence of the excitation and depletion beam intensities and dye concentrations on the depletion efficiency are studied in detail. The results indicate the following: (1) The highest depletion occurs for the 100 μM Coumarin 102 solution, with a 1.4 μW excitation beam and a 115.3 mW depletion beam. (2) The minimum saturation intensity (Is) of STED, that is 13 MW cm^-2 , is observed when the Coumarin 102 solution concentration is 10 μM. (3) Is values calculated directly from the depletion power derived with the cross-sectional area due to the full-width-at-half-maximum (FWHM) of the depletion beam show poor accuracy, where Is may be overestimated. Thus, a correction factor for the cross-sectional area is proposed. We also find that Is is not exactly constant for a fixed excitation beam power and dye concentration. This trend indicates that the conventional suppression function η(x)=e- ln (2)ISTED(x)/Is derived from picosecond STED may cause errors in evaluating the depletion process in CW STED microscopy.