Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores

For decades, the power of far-field fluorescence microscopy has been hampered by the diffraction resolution limit,1,2 d) λ/(2NA)> 200 nm,3,4 where λ denotes the light wavelength and NA the numerical aperture of the lens. Identical fluorophores closer than d cannot be discerned because their signals cannot be separated by the detector. However, in the recent past, it has been demonstrated that the diffraction limit can be fundamentally overcome5 by modulating or switching the ability of the dye to emit fluorescence, causing adjacent objects to be registered sequentially. To this end, stimulated-emission depletion (STED) microscopy5,6 and its deriva-tives modulate the fluorescence capability of dye ensembles using defined spatial patterns of light, whereas the techniques known as (f)PALM,7,8 (d)STORM,9,10 and related concepts11 involve switch-ing individual fluorophores stochastically in space followed by mathematical localization of their coordinates. Hence, all of these concepts harness a molecular mechanism that keeps the dy