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.

About SciLifeLab

Our lab is located in the Science for Life Laboratory (SciLifeLab), a national center for molecular biosciences with focus on health and environmental research. SciLifeLab has been created by the coordinated effort of four universities in Stockholm and Uppsala: Stockholm University, Karolinska Institutet, KTH Royal Institute of Technology and Uppsala University.

Open Positions

We are looking for self-motivated and curiosity-driven candidates with an expertise in physics, chemistry or biology, who are looking to make original contributions to the field of super-resolution microscopy. The candidates will work on collaborative projects investigating the nanoscale organization and dynamics of proteins in living neurons and brain tissue with our cutting edge microscopy technology. We offer an outstanding scientific environment and a vibrant working climate with individual freedom and various possibilities for professional development. The work will be funded by the European Union within the ERC project “MoNaLISA”.

Get in Touch

    Ilaria Testa, PhD.
    Assistant Professor
    KTH Royal Institute of Technology
    Dept. of Applied Physics
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    Science for Life Laboratory
    Tomtebodavägen 23A
    171 65 Stockholm