Strategies to maximize the performance of a STED microscope

In stimulated emission depletion (STED) microscopy, the spatial resolution scales as the inverse square root of the STED beam's intensity. However, to fully exploit the maximum effective resolution achievable for a given STED beam's intensity, several experimental precautions have to be considered. We focus our attention on the temporal alignment between the excitation and STED pulses and the polarization state of the STED beam. We present a simple theoretical framework that help to explain their influence on the performance of a STED microscope and we validate the results by imaging calibration and biological samples with a custom made STED architecture based on a supercontinuum laser source. We also highlight the advantages of using time gating detection in terms of temporal alignment.