The key to overcome the diffraction barrier with freely propagating light waves is to use the properties of the fluorescent markers such that they are transferred between a bright, detectable state and a dark, non-detectable state or vice versa.
This is done in such a way that the region in which the markers can exist in their bright state is spatially confined.
The STED (STimulated Emission Depletion) principle uses stimulated emission to “switch” the fluorescent markers from their (bright) excited electronic state S1 to the (dark) electronic ground state S0.
In order to confine the region in which the markers are allowed to fluoresce beyond the diffraction limit, the excitation focus is superimposed by a donut-shaped STED focus. The former excites an ensemble of markers, while the latter switches these molecules off so rapidly by stimulated emission such that they do not have time to emit a fluorescence photon.
Only in the very center of the donut, at which the STED intensity is zero, the fluorescence emission is still allowed.
The STED image is gained by scanning the fluorescent spot, whose extent depends on the STED intensity and can be theoretically decreased arbitrarily, over the sample.
Themes within STED microscopy:
- Website of Department of NanoBiophotonics at the MPI for biophysical chemistry:
- S. W. Hell (2007):
"Nanoskopie mit fokussiertem Licht"
Physik Journal 6 (12), 47 - 53
- S. W. Hell (2008):
"Microscopy and its focal switch"
Nature Meth. 6 (1), 24 - 32, Perspective, Special Feature, see Method of the year 2008
- S. W. Hell (2009):
"Far-Field Optical Nanoscopy"
In: Single Molecule Spectroscopy in Chemistry, Physics and Biology. Springer (Berlin, Germany), 365 - 398
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