Stimulated Emission Depletionits
STED microsocopy uses a second, red-shifted light beam. All molecules irradiated with this STED light are confined to the ground state (off-mode). The key is to modify the STED-beam in such a way that it has a tiny hole in the center where its intensity is zero, for example by giving it the well-known STED-“donut” shape. This way, all molecules except the ones in the center of the donut stay dark temporarily. Any fluorescence that is detected must necessarily come from this sub-diffraction-sized spot – their location is now known more precisely. The diffraction barrier is broken.
Below is a Jablonski diagram of the STED process of a fluorescent molecule. After optical excitation (1) from the ground state S0 to the first excited state S1 two ways are possible to return into the ground state: in case the molecule is in an area with STED photons, the electron gets stimulated down into the ground state and no fluorescence occurs (2). If the molecule is in the center of the donut, it returns to the ground state under emission of a fluorescence photon (3).
To implement the STED technique, the microscope design needs to consider a second focused 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. The phaseplate is responsible for converting the plain STED beam into a donut shape.
The complete image is created by scanning the overlayed focus of both beams through the sample. At each position, the signal from the very center of the focus is detected.
STED microscopy was the first technique to abandon 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 excitation wavelength and I denotes STED intensity. NA is the numerical aperture of the objective lens and I_sat denotes how well the dye reacts to STED photons.
Strictly speaking, the detector only needs to see one photon to identify an individual molecule (or cluster) with nanoscale resolution. No mathematical algorithms are needed to render an image below the resolution barrier, STED is pure physics! Once molecues are separated, they can be localized even better (below).