STED microscopy: Exploring fluorescence lifetime gradients for super-resolution at reduced illumination intensities

The 21st century has opened with the development of several strategies to push the spatial resolution of far-field fluorescence microscopy beyond the diffraction limit. Among all these super-resolved techniques, stimulated emission depletion (STED) microscopy stands out for its general principles and its fast acquisition capability. However, the application of STED microscopy to live-cell imaging has been limited by the typically high illuminationpowers required by early implementations of the technique. One way to preserve the effective resolution of a STED microscope, but with a significant reduction of the illumination intensity, is by using the nanosecond fluorescence dynamics information contained in a time-resolved STED experiment. Two different implementations exploring this idea have been demonstrated so far, respectively the so-called gated-STED (g-STED) microscopy and Separation of Photons by Lifetime Tuning (SPLIT)-STED microscopy. Here we discuss the common principle behind the two methods and the benefits and major differences between the two implementations.