Photon-separation to enhance the spatial resolution of 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, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method 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 the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.