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

In 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.