RESOLFT

The RESOLFT microscopy (English reversible saturable optical ( flurorescence ) transitions, dt, reversible saturable optical (fluorescence ) transitions ') is a group of light- microscopic methods, in which one obtains particularly sharp images. Despite the use of conventional lenses and diffracted beams resolution is far beyond the diffraction limit get down to the molecular scale.

In a conventional light microscope, no details can be distinguished, which are closer together than approximately 200 nm, this restriction is due to the wave nature of light. In conventional light microscopes this resolution limit is essentially determined by the wavelength of light used and the numerical aperture. The RESOLFT microscopy overcomes this limit by switching the dyes temporarily in a state in which they are not able to respond with a signal after illumination (fluorescence ).

• 2.1 STED microscopy
• 2.2 GSD microscopy
• SPEM 2.3 and SSIM
• 2.4 RESOLFT microscopy with switchable proteins
• 2.5 RESOLFT microscopy with switchable organic dyes
• 2.6 generalizations

Principle of operation

The RESOLFT microscopy is a variation of light microscopy. It overcomes the diffraction limit by which records details of a preparation, which normally are too close together to be resolved one by one. Thus the principle of STED and GSD Miksroskopie is generalized to arbitrary types of molecules that can be switched between two distinct states A and B reversible. The switching of the dye molecules in at least one of the two states (eg from the ground state A in the dark state B) can be induced by light.

The specimens are to be examined, marked with special molecules, usually fluorescent dyes. The RESOLFT microscopy using optically driven, distinguishable states in the marker molecules. The molecules are thereby switched back and forth between at least two states: a signaling ( bright ) state A and a dark state B. The switching of the (dye ) molecules in at least one of the two states (eg to state B ) can be induced by light.

The preparation is thereby inhomogeneous illuminated. The illumination intensity is at least one predefined location very low, ideally zero (ie completely dark). Only in these dark places, the molecules are therefore not brought into the state B and A. This area remain in can then be very small ( much smaller than the classical diffraction limit ) make ( see below). Upon detection of the signal ( usually fluorescent light ) is now known that there can be only in this small region. By moving the "A- frame " on the preparation and assembly of the sub-images ( scanning) can therefore obtain images with the higher resolution of the "A- frame ".

The passage of the marker molecules in the other areas of the image back to the state A, for example, spontaneously or by light of other wavelengths. The marker molecules have to repeatedly make switch back and forth between the states for A and B to always keep in targeted locations to state A and in the neighboring areas of the state B. The molecules need not necessarily be connected to the signaling state in the small selected area. Also a negative imaging is possible in which you get no signal from just the small area. In this case, a mathematical post-processing of the images is necessary to obtain a positive image.

Reduction below the diffraction limit

This can be achieved because despite the diffraction limit of the area where the light intensity is so low that the molecules remain in state A, can be made arbitrarily small (see figure):

To understand this principle, we make two assumptions:

If the preparation is now illuminated with low intensity (left figure), is the area in which the molecules are in state A (marked in green in the figure ) is relatively large. Already, by increasing the intensity (that is, without changing the shape of the illumination profile is changed), the area in which the intensity is low, smaller (right image) is. Consequently, it is also the area in which the molecules are in state A, small (marked in green in the figure). Thus, the fluorescence signal comes only from a very small area and it will get clearer pictures.

Variants

For switching the marker molecules different processes are used, which are described below. All methods have in common that the marker at least between two states is switched back and forth: a signaling ( bright ) state A and a dark state B.

STED microscopy

( See also the article on the STED microscope).

In STED microscopy (English: Stimulated Emission Depletion Microscopy), a fluorescent dye in A between its electronic ground state and the excited state swing back and forth and fluoresce it. In B, the dye is held by stimulated emission permanently in its ground state. So there are two configurations of the fluorescent dyes: in A can fluoresce in B and not the conditions for RESOLFT are available.

GSD microscopy

Even with the GSD microscopy (English Ground State Depletion Microscopy, dt " Grundzustandsentvölkerungsmikroskopie " ) fluorescent dyes are used as markers. The dye may be in the bright state A between the ground state and the excited state swing back and forth and fluoresce it. For the dark state is the ground state of the molecule B is depopulated: The molecule is excited to a durable state from which there is no fluorescence. As long as the molecule is in the long-lived dark state, it is not in the ground state is available, so it can not be excited and therefore not fluoresce. The return to the light state A takes place spontaneously. Often it is in the long-lived state is a so-called triplet state. Of nitrogen -vacancy centers in diamond a resolution of up to 7.8 nm was achieved. Compared with a conventional light microscope ( confocal ) recording the gain in resolution is particularly evident ( see Fig.)

SPEM and SSIM

SPEM (English: saturated pattern excitation microscopy) and SSIM (English: saturated structured illumination microscopy) are RESOLFT method, the first record negative images and use a mathematical image reconstruction. The ground state occurs here at the location of the dark state B and the first excited state is the bright state A.

RESOLFT microscopy with switchable proteins

Some fluorescent proteins may be turned on and off by the light of appropriate wavelength and thus used for RESOLFT microscopy. By irradiation with light, they change their structure. Only one of these structures, the protein capable of fluorescence. By this light-induced structural change of these proteins can thus be switched from a state A, a state B, one of which is fluorescent only one. The transition from the state B to the state A takes place either spontaneously or by light. For the switching of proteins, only very low light intensities are needed ( a few watts per square centimeter) in comparison to the method STED and GSD.

RESOLFT microscopy with switchable organic dyes

As well as in some proteins, structural changes can be induced by light in particular organic dyes. The fluorescence ability of such dyes can be just as in the proteins by light on and off. Also only relatively low intensities are needed (several hundred Watts per square centimeter).

Generalizations

The two states A and B must be distinct, but it need not necessarily be involved fluorescence. Switching between an absorbent and a non- absorbing state or a scattering and non- scattering state would also be possible.

Swell

• Stefan W. Hell: Microscopy and its focal switch. In: Nature Methods. Vol 6, No 1, 2009, pp. 24-32, doi: 10.1038/nmeth.1291.
• Stefan W. Hell: Far - Field Optical Nanoscopy. In: Science. Vol 316, 2007, pp. 1153-1158, doi: 10.1126/science.1137395.