Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.
In a stimulated emission depletion (STED) microscope the region from which a fluorophore can spontaneously emit shrinks with the continued STED beam action after the excitation event. This fact has been recently used to implement a versatile, simple and cheap STED microscope that uses a pulsed excitation beam, a STED beam running in continuous-wave (CW) and a time-gated detection: By collecting only the delayed (with respect to the excitation events) fluorescence, the STED beam intensity needed for obtaining a certain spatial resolution strongly reduces, which is fundamental to increase live cell imaging compatibility. This new STED microscopy implementation, namely gated CW-STED, is in essence limited (only) by the reduction of the signal associated with the time-gated detection. Here we show the recent advances in gated CW-STED microscopy and related methods. We show that the time-gated detection can be substituted by more efficient computational methods when the arrival-times of all fluorescence photons are provided.