Stimulated Emission Depletion microscopy, or STED microscopy, is a fluorescence microscopy technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field optical microscopes. Therefore, it is also described as a form of super-resolution microscopy.
A confocal laser scanning microscope uses a focused laser beam to illuminate a small part of the sample being observed. The laser is tuned to a frequency that excites fluorescence from dye molecules in the sample, and light from the small region being excited is subsequently observed by a detector. The resolution of such a microscope is limited to the spot size to which the excitation spot can be focused. This size depends on system parameters, but is limited by approximately half the wavelength of the light used. Nearby structures in the focal plane with a distance smaller than about 200 nm cannot be resolved. The resolution along the optical axis (depth resolution) is notably worsened, around 500 nm even with objective lenses with high numerical aperture. Within the STED microscope, the diffraction limit is surmounted by targeted strong de-excitation of dye molecules, in effect switching them off. A resolution of up to 5.8 nm could be achieved.[1]
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Stimulated Emission Depletion microscopy uses fluorescent dyes to label specific sites of a sample. Such dyes can be excited by light of certain wavelengths. During excitation, a photon of this light can be absorbed by a dye molecule, and the molecule will be in an excited electronic state. From the excited state, the molecule can spontaneously relax to the ground state after a short time (typically some nanoseconds). During this decay, a fluorescence photon of red-shifted (greater) wavelength is emitted. By using color filters, the fluorescent light can be separated from the excitation light.
Instead of spontaneous relaxation and fluorescence emission, a molecule can also return to its ground state by stimulated emission. If an excited dye molecule is irradiated with light of similar wavelength compared to the fluorescence light, it can immediately return to the ground state and emits a photon of exactly the same wavelength and momentum of the light used. Furthermore, the molecule is prevented from the spontaneous emission of a fluorescence photon after stimulated emission. Fluorescent dyes can, therefore, be switched off by additional irradiation of a red-shifted 'de-excitation' beam. The light originating from the spontaneous decay and from the stimulated emission can also be spectrally separated from the fluorescent light by using appropriate color filters.
The minimal size of a focused light spot is limited by diffraction to about half the wavelength used. Laser-scanning microscopes can therefore not decrease the size of the excitation spot for fluorescent dyes further than approximately 200 nm, half the wavelength of blue light. In a STED microscope the excited molecules in the outer rim of the excitation spot are additionally switched off by stimulated emission. Therefore a second, red-shifted 'de-excitation' laser beam is focused into the sample whose wavefront is altered so that a ring-like intensity profile is achieved.[2] While this light distribution with a dark spot in its center is itself diffraction-limited, it features at least some intensity near the focus and is zero only at the very center. Therefore, using intense depletion light causes almost all of the excited molecules to return to the ground state, leaving only the region of the sample very close to the center of the excitation spot excited. Fluorescence from the remaining excited dye molecules is then detected by the microscope.
The size of the spot where molecules are still allowed to fluoresce gets smaller with increasing intensity of the de-excitation light. This size corresponds to the achievable resolution. Therefore the resolution is controlled by the brightness of the de-excitation beam. The resolution can become much better than the diffraction-limit, indeed an arbitrary good resolution is possible provided that one can apply such a very high de-excitation beam intensity.
By switching-off the excited molecules in a saturated manner, only a fraction of molecules in an area much smaller than the original focused excitation spot can fluoresce. Therefore structures that are smaller than the diffraction-limit can be made visible with STED. It was the first implementation of a more general concept known as RESOLFT, wherein all saturable transitions between two optically distinguishable states of a dye molecule are used, not only stimulated emission. One of these methods proposed early on is Ground State Depletion microscopy,[3] which uses an excitation pulse to boost off-axis ground-state molecules to convert to a long-lived, non-fluorescing higher-energy state, leaving, again, only the molecules near the center of the focus able to fluoresce.
An important problem for all optical microscopy techniques is the lack of meaningful contrast in samples like living cells. For this, fluorescent molecules that are linked to the objects of interest via anti-bodies or genetically encoding are used. For example, only the nucleus of a cell can be specifically labeled and recorded with a fluorescent dye. However, these dyes work often with visible light, and the images are therefore subject to the diffraction-limit; small structures cannot be resolved. With STED microscopy, even living cells can be recorded at a much higher resolution[4]. Electron microscopy, which also has a very high resolution, is not live-cell-compatible, and needs vacuum and thin cutting of the sample. Near-field scanning optical microscopes also show increased resolution but are, unlike STED, bound to the investigation of surfaces.
Also highly dynamic processes have been observed by STED[5].
A commercial STED Microscope is produced by microscope manufacturer Leica Microsystems.
The idea was published in 1994 by Stefan Hell.[6] and a first experimental realization was demonstrated in 1999[7].
Since then, the number of applications as well as the achievable resolution have increased significantly. In 2006 imaging biological macromolecules a resolution improvement over confocal laser scanning microscopy of up to 12-fold have been reported.[8]. As of 2009 the highest resolution achieved is 5.8 nm on color centers of nanocrystals as the fluorescing unit.[1]
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