Evaluation of saturation intensity based on the FWHM of CW stimulated emission depletion microscopy

We propose a numerical method to evaluate the saturation intensity of fluorescence, which is a key parameter in determining the spatial resolution of a stimulated emission depletion (STED) microscopy system. The saturation intensities of four different continuous-wave (CW) STED systems reported from the studies of previous researchers (Willig et al., Nature Methods, 2007, 4, 915-918; Hein et al., PNAS, 2008, 105, 14271-14276; Hotta et al., JACS, 2010, 132, 5021-5023) have been calculated. It is found that pTDI fluorescent dye with a 633-nm excitation beam and a 780-nm depletion beam can provide the smallest saturation intensity (or the best depletion effect under the same depletion power) in all four cases. Based on the saturation intensity, we revisit the influence of wavefront modulation on the resolution improvement of the STED system. It is found that by applying azimuthally polarized light, the lateral resolution of the STED system could be increased to 26 nm with an improvement of up to 67%.