Role of the Pico- Nano-Second Temporal Dimension in STED Microscopy

n the last decades, several techniques have been developed to push the spatial resolution of far-field fluorescence microscopy beyond the diffraction limit. Stimulated emission depletion (STED) microscopy is a super-resolution technique in which the targeted switching off of the fluorophores by a secondary laser beam results in an effective increase in optical resolution. However, to fully exploit the maximum performances of a STED microscope (effective spatial resolution achievable for a given STED beam’s intensity, versatility, live-cell imaging capability, etc.) several experimental precautions have to be considered. In this respect, the temporal dimension (at the pico- and nanosecond scale) has often a central role on the overall efficiency and versatility of a STED microscope, working in pulsed or continuous-wave mode. In pulsed STED, temporal alignment between the excitation and STED pulses has direct consequences on the maximum spatial resolution achievable by the STED microscope. In a specific pulsed STED implementation, called single wavelength two-photon excitation STED, the modulation of the temporal width of the pulse results in the use of the very same laser for excitation and depletion of the fluorophores. In continuous-wave (CW)-STED, the analysis of nanosecond fluorescence dynamics allows to preserve the effective resolution of a STED microscope, but with a significant reduction of the illumination intensity. In this respect, we discuss two different approaches for the analysis of nanosecond dynamics in CW-STED images, namely the so-called gated-STED microscopy and Separation of Photons by LIfetime Tuning (SPLIT)-STED microscopy. Overall, these examples show that concepts developed in time-resolved fluorescence spectroscopy are important for the advancement of optical super-resolution microscopy.