Photon-separation to enhance the spatial resolution in pulsed STED microscopy

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, whose intensity is directly related to the achievable resolution. Despite achieving nanometer resolution, much effort in recent years has been devoted to reduce the STED beam intensity because it may lead to photo-damaging effects. Exploring the temporal dynamics of the detected fluorescence photons and accessing the encoded spatial information has proven to be a powerful strategy, and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT technique uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on STED beam running in continuous wave (CW-STED microscopy). Here, we extend it to architectures based on pulsed STED beam (pSTED microscopy). We show, through simulated and experimental data, that the SPLIT-pSTED method reduces the detection volume of the pSTED microscope without significantly reducing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.