Have you ever asked yourself, how STED works? What’s the difference between STED, STORM, and MINFLUX? What is “targeted readout” and “stochastic readout”? How exactly does RESOLFT work? Read on to find out.
If individual marker molecules (black dots below) are closer than the diffraction barrier they cannot be imaged separately – their individual blurred images overlap and all you see is an unsharp, hazy soup without any details under 200 nm.
Due to diffraction, the area illuminated by a focused excitation laser (green, below) is always larger than half the wavelength of the light used. Every molecule located within this area emits light if there’s more than one there is no way they can be distinguished.
All fluorophors adjacent to a sub-diffraction-sized area are temporarily kept dark, so that they can be registered separately. This way, their blurred images do not overlap they can be recorded sequentially. The basic receipt for superresolution is indeed ingeniously simple – introduce a controllable on/off transition of the dye label.
Consequently, the dye is the key for nanoscale resolution. abberior is specialized in designing dyes with on/off transitions that can be controlled via a second light source.