STED microscopy
A STED microscope ( STED = Stimulated Emission Depletion) is a fluorescence microscope whose resolution is not diffraction limited and operates on the principle RESOLFT. It was founded in 1994 described theoretically by Stefan Hell and experimentally realized by him in 1999. Since then, there will be further developed among others in his group at the Max Planck Institute for Biophysical Chemistry in Göttingen.
Because of diffraction, the resolution of conventional far-field microscopes is limited. It can therefore not resolve details smaller than about half the wavelength of the light used. Confocal microscopes can therefore dissolve adjacent structures only up to a size of about 200 nm. The resolution for consecutive objects ( depth resolution ) is clearly worse. The STED microscope, the resolution is increased beyond the diffraction limit, by selectively turning off of fluorescent dyes. It has already been shown a resolution of 2.4 nm ( lateral).
The STED microscope and the group led by Stefan Hell were honored for their results in 2006 with the German Future Prize.
Basics
A STED microscope is based on fluorescence laser scanning microscopes. To better understand the operating principle, it is necessary to address the most important aspects of fluorescence, stimulated emission and laser scanning microscopy.
Fluorescence and Stimulated Emission
In STED microscopy, called fluorescent dyes are used to highlight specific areas of a specimen. Such dyes may be " excited " by the light of certain wavelengths ( colors): They absorb a photon and go on to a more energetic state. From this state, they can return to the ground state after a short time spontaneously by emitting a photon of longer wavelength (different color). This spontaneous emission light is called fluorescence. By a suitable color filter, the fluorescent light can be separated from the excitation light.
An excited dye molecule can return except by fluorescence by stimulated emission back to the ground state. This occurs when the excited dye molecule with light of approximately the same wavelength as that of the fluorescence light is irradiated. The excited dye molecule can be exactly for immediate transition to the ground state by emitting a photon stimulates the same wavelength. Spontaneous fluorescent light can not be sent then that molecule is no longer even in the excited state. Fluorescent molecules can therefore be disabled by Stimulated emission: When they are stimulated emission of light, they can give no spontaneous fluorescence light thereafter. Spontaneous fluorescent light and light from the stimulated emission can be separated, for example by color filters.
Fluorescence laser scanning microscopy
For the investigation in a fluorescence microscope, fluorescent dyes are brought to certain points of the preparation to be examined. If the preparation is now illuminated with light of a suitable wavelength, the dyes are excited to fluorescence, and gives an image of the dye distribution in the specimen. In the laser scanning microscope, the entire specimen is not illuminated at once, but a laser beam ( " excitation beam " ) is only focused to a small point in the preparation. The excited fluorescence is detected in this area. By moving (scanning, grids ) of the focus on the preparation an image of the colored areas will be created pointwise. The size of the focus determines the maximum fineness of detail that can be just resolved in the preparation ( perceived separately ). Because of diffraction, the focus can not be chosen arbitrarily small. A laser beam can not be to a spot smaller than about focus its half wavelength.
Operating principle of the STED microscope
A STED microscope with better resolution than a conventional laser scanning microscope is possible: The area is emitted from the fluorescence is in this case made significantly smaller than the area that is illuminated by the laser beam. This is achieved by selectively turning off of the dye molecules in the outer region of the focus. For this purpose, the preparation is not only with the focused excitation beam illuminated (left image), but at the same time with a second laser beam, the " Ausschaltestrahl ". This Ausschaltestrahl gives you an annular profile in focus ( middle image). In the middle, ie, where the excitation beam has its maximum brightness, the Ausschaltestrahl is completely dark. The Ausschaltestrahl so does not affect the fluorescent dyes in the middle. He switched the fluorescent dyes in the outer region of the excitation focus by stimulated emission (see above); the dye molecules in the outer region remains dark, even though they are illuminated by the excitation laser. It is therefore only light up the dye molecules exactly from the center ( right). The minimum diameter of the excitation beam is diffraction limited, although just like the central dark field of Ausschaltestrahls. However, just a few photons of Ausschaltestrahls to stimulate the emission of a larger number of excited states; In addition, the intensity of the Ausschaltestrahls can be set higher than that of the excitation beam. Characterized the non- switched-off central area is much smaller than the illuminated area of the excitation laser (see line profiles on the right). When scanning the specimen thus we recorded each a luminous spot, which is much smaller than in a normal laser scanning microscope. Therefore, one can resolve finer details. To get a complete picture, the preparation is scanned point by point.
The size of the resulting light spot decreases with increasing intensity of Ausschaltestrahls from more and more. This means that the resolution rises all the more, the brighter the Ausschaltestrahl; the achievable resolution is not limited in principle. Before the invention of STED microscopy has been the problem that the excitation beam due to the Abbe diffraction limit can not be focused as small as desired. So you always encourages all molecules that is currently in focus, and therefore can not decide which of the fluorescent molecule is straight. Could therefore structures that are smaller than the dimension of the laser focus, can not be distinguished.
Applications
A major problem of any light microscopic technique is the lack of contrast of cell components. For a long time therefore we used fluorescent molecules that can be attached, for example by genetic engineering methods or by means of antibodies selectively to certain molecules of a cell. You can grow as dyes only in mitochondria. If you light now a place of the thus prepared cell with a focused laser beam from where it gets fluorescence, as were dye molecules and thus mitochondria at exactly this point. To get a complete picture, the preparation is scanned point by point. In a STED microscope can examine all the preparations that can be labeled with fluorescent dyes. Unlike electron microscopes, no vacuum and no thin sections are required. Therefore, even living cells can be observed.
In contrast to scanning probe microscopy STED microscopy is a far-field technique. It is therefore not limited to the study of surfaces. Example, one can also examine the inside of cells. The observation of fast dynamic processes is possible.
Leica Microsystems manufactures since 2007, commercial versions of the STED microscope.