Comparison of three forms of fluorescence microscopy. In conventional fluorescence microscopy (top), a single laser (shown here in blue) is used to excite green fluorescent protein (GFP) or some other fluorophore, and the resulting fluorescence is detected. The resolution is determined by the size of region in which the GFP is excited (shown in green) and corresponds to the diffraction-limited laser focus size. In stimulated emission depletion (STED) nanoscopy (middle), a second ring-shaped laser focus (shown here in orange) is used to transfer excited GFP molecules at the periphery of the excitation focus back to the ground state without fluorescing: the size of the region in which the GFP can still fluoresce is smaller than in conventional fluorescence microscopy and is no longer limited by the laser focus size. In RESOLFT nanoscopy with rsEGFP2 (bottom), inactive fluorophores (shown as black molecular structures) are first activated by a violet laser focus. A second blue ring-shaped laser focus then continuously excites activated fluorophores until they have switched back to the inactive state. After these two steps, a conventional blue laser focus is used to illuminate the remaining active molecules and the resulting fluorescence is detected. Similar to STED nanoscopy, the size of the region in which rsEGFP2 can still fluoresce is not limited by diffraction. Typical wavelengths, laser intensities, and illumination times are shown for each laser. The period during which fluorescence is detected by all three methods is comparable (∼1 to 10 μs). Note that GFPs are not shown to scale. EGFP is enhanced GFP.