STED makes use of stimulated emission by introducing a second, red-shifted light beam. All molecules irradiated with this STED light are captured inthe ground state (off-mode).
The following depicts a Jablonski diagram of the STED process of a fluorescent molecule.
After optical excitation from the ground state S0 to the first excited state S1 two ways are possible to return into the ground state: (1) In case of STED the electron gets stimulated down into the ground state and no fluorescence occurs (2) Without STED the molecule returns under emission of a fluorescence photon into the ground state (3).
To implement the STED technique the microscope design needs to consider a second light beam (STED light) besides the excitation beam. Molecules in areas subject to STED light above the saturation threshhold are forced into the off state.
A STED microscope creates the image via predetermined scanning of the overlayed focus of both beams through the sample. At each recorded position the signal from the very center of the focus is detected.
The STED technique was the first technique that abandons the diffraction barrier in optical microscopy. STED features theoretically unlimited resolution, which can be expressed by a 'modified' Abbe-equation:
where Δx denotes the smallest feature in space that is resolvable, λ denotes the excitation wavelength and I denotes the applied STED depleting intensity.
Strictly speaking, the detector only needs to see at minimum one photon to identify an individual molecule (clusters) with a nanoscale resolution. No calculation algorithms<//b> are needed to record an image below the resolution barrier.