Efficient two-photon excitation stimulated emission depletion nanoscope exploiting spatiotemporal information

Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.