STED, STimulated Emission Depletion
In STED microscopy the diffraction barrier is bypassed by controlling the switching of molecules in a deterministic way, point by point with the superimposition of a light pattern featuring local zero of intensities. STED microscopy uses a dedicated ‘STED’ beam to modulate the fluorescence capability of fluorophores residing closer than the diffraction barrier in order to make them distinguishable. In a typical implementation, a focused laser beam is used to raise the fluorophores to their excited state – as in confocal microscopy. However, the excitation spot is superimposed with the ‘STED beam’, which is typically ring-shaped. The role of this beam is to transiently silence the emission of the fluorophore by the phenomenon of stimulated emission. Stimulated emission means that the fluorophores are instantly sent to the ground state before they are capable of emitting a fluorescent photon spontaneously. The silencing takes place because the wavelength and intensity of the STED beam are chosen so as to take away the majority of the excitation energy in a copy photon of the STED beam, which is discarded.
Because at least a single de-exciting photon must be available within the lifetime (τ ≈ 1–5 ns) of the fluorescent molecular state, the intensity of the focal STED beam must exceed the threshold Is = Cτ−1 with C accounting for the probability of a STED beam photon to interact with the fluorophore.
Only fluorophores residing in the direct vicinity of the zero-intensity minimum of the STED focus can effectively assume the fluorescent state and hence contribute to the fluorescence signal. If Δ is the conventional resolution (~200-300nm), the diameter of this area is given by
with Im (typically twice greater than Is) denoting the intensity at the ring crest. Hence, features that are (just slightly) more apart than d < λ cannot fluoresce at the same time even when simultaneously illuminated by excitation light. By scanning the superimposed excitation and ring-shaped beam focal spots jointly across the specimen, features that are closer than the diffraction barrier assume the fluorescent state sequentially and are thus readily separated. STED is a direct and immediate imaging method because it immediately achieves high spatial resolution (down to 15-20nm) without any requirements of image reconstruction. It is this feature that made STED the method of choice for many applications in cells and even in living mice.