RESOLFT microscopy

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The resolft ( English reversible saturable optical linear (fluorescence) transitions , dt., Reversible saturable optical (fluorescence) transitions') is a group of light microscopic method wherein the obtained especially sharp images. Despite the use of conventional objectives and diffracted beams, a resolution is obtained far beyond the diffraction limit down to the molecular scale.

A conventional light microscope cannot distinguish details that are closer together than about 200 nm. This limitation is due to the wave nature of the light. In conventional light microscopes, this resolution limit is essentially determined by the wavelength of the light used and the numerical aperture . RESOLFT microscopy overcomes this limit by temporarily switching the dyes into a state in which they are unable to respond with a (fluorescent) signal after illumination.

Working principle

RESOLFT microscopy is a variant of light microscopy . It overcomes the diffraction limit by taking in the details of a specimen one after the other, which are normally too close together to be resolved. The principle of STED and GSD microscopy is thus generalized to any type of molecule that can be reversibly switched between two distinguishable states A and B. The switching of the dye molecules into at least one of the two states (e.g. from the ground state A to the dark state B) can be brought about by light.

The preparations to be examined are marked with special molecules, mostly fluorescent dyes . RESOLFT microscopy uses optically driven, distinguishable states in the marker molecules. The molecules are switched back and forth between at least two states: a signaling (light) state A and a dark state B. Switching the (dye) molecules to at least one of the two states (e.g. into state B) can be brought about by light.

The specimen is illuminated inhomogeneously. The lighting intensity is very low at at least one predefined point, ideally zero (i.e. completely dark). It is only in these dark areas that the molecules are not brought into state B and remain in A. This area can then be made very small (much smaller than the classic diffraction limit) (see below). When the signal (mostly fluorescent light) is detected, it is now known that it can only come from this small area. By moving the "A-area" over the preparation and combining the partial images ( scanning ), images with the higher resolution of the "A-area" can be obtained.

The transition of the marker molecules from the other image areas back to state A can take place, for example, spontaneously or through light of a different wavelength. The marker molecules have to be able to switch back and forth between states A and B several times in order to always retain state A at specific points and state B in the neighboring areas. The molecules do not necessarily have to be switched to the signaling state in the small selected area. Negative imaging is also possible, in which one does not get a signal from the small area. In this case, mathematical post-processing of the images is necessary in order to obtain a positive image.

Reduction below the diffraction limit

RESOLFT principle: the specimen is illuminated inhomogeneously (red line). The lighting intensity is zero at one point. In the area around this zero point the intensity is below the threshold (blue line) for switching the marker molecules to the dark state B. Only in the area around the zero point (green marked area) do the molecules remain in the signaling state A.
Left: At If the lighting intensity is low, the (green) area below the threshold is relatively large.
Right: By increasing the intensity (without changing the lighting profile), the area below the threshold becomes much smaller.

This can be achieved because, despite the diffraction limit, the area in which the illumination intensity is so low that the molecules still remain in state A can be made as small as desired (see figure):

To understand the principle, let's make two assumptions:

  1. The specimen is illuminated in such a way that the intensity is zero at one point (red line in the figure). Such lighting can be used e.g. B. realize by interference effects .
  2. At low intensities (lower than the intensity marked by the blue line in the figure), the marker molecules are in the (light) state A. At higher intensities, the molecules are in the (dark) state B. However, this is a simplification - the transitions are usually not that abrupt.

If the preparation is now illuminated with low intensity (left fig.), The area in which the molecules are in state A (marked green in the fig.) Is relatively large. Just by increasing the intensity (i.e. without changing the shape of the lighting profile), the area in which the intensity is low becomes smaller (right illustration). As a result, the area in which the molecules are in state A also becomes smaller (marked in green in the figure). Thus the fluorescence signal only comes from a very small area and sharper images are obtained.

variants

Various processes are used to switch the marker molecules, which are described below. What all methods have in common is that the marker is switched back and forth between at least two states: a signaling (light) state A and a dark state B.

STED microscopy

(See also the article on the STED microscope ).

In STED microscopy ( Stimulated Emission Depletion Microscopy ), a fluorescent dye in A can oscillate back and forth between its electronic ground state and the excited state and thereby fluoresce. In B the dye is permanently held in its basic state by stimulated emission . There are two configurations of fluorescent dyes: they can fluoresce in A, not in B and the requirements for RESOLFT are present.

GSD microscopy

Resolution comparison between conventional (confocal) microscopy and GSD microscopy. Left: conventional recording of flaws in diamonds. The individual imperfections cannot be seen separately. Right: GSD image of the same location. The individual imperfections are very clearly separated from each other. The apparent size of the individual defects, which is determined by the resolution of the microscope, is only 15 nm.

Fluorescent dyes are also used as markers in GSD microscopy ( Ground State Depletion Microscopy ) . In the light state A, the dye can oscillate back and forth between the ground state and the excited state and fluoresce in the process. For the dark state B, the ground state of the molecule is depopulated: the molecule is excited into a long-lived state from which no fluorescence occurs. As long as the molecule is in the long-lived dark state, it is not available in the ground state, i.e. it cannot be excited and accordingly cannot fluoresce. The return to the light state A occurs spontaneously. The long-lived state is often a so-called triplet state . A resolution of up to 7.8 nm was achieved at nitrogen vacancy centers in diamonds. In comparison with a conventional light microscope (confocal) image, the gain in resolution is particularly clear (see Fig.).

SPEM and SSIM

SPEM ( saturated pattern excitation microscopy ) and SSIM ( saturated structured illumination microscopy ) are RESOLFT processes that initially record negative images and use mathematical image reconstruction. The ground state takes the place of the dark state B and the first excited state is the light state A.

RESOLFT microscopy with switchable proteins

Some fluorescent proteins can be switched on and off by light of a suitable wavelength and thus used for RESOLFT microscopy. When exposed to light, they change their structure. The protein is only capable of fluorescence in one of these structures. Through this light-induced structural change, these proteins can be switched from a state A to a state B, only one of which is fluorescent. The transition from state B back to state A occurs either spontaneously or through light. In comparison to the STED and GSD methods, only very low light intensities are required to switch the proteins (a few watts per square centimeter). In combination with 4Pi microscopy ( 4Pi microscope ), images with isotropic resolution (<40 nm) in living cells and low light intensities were created for the first time in 2016.

RESOLFT microscopy with switchable organic dyes

As in some proteins, structural changes in certain organic dyes can also be induced by light. The fluorescence ability of such dyes can be switched on and off by light, just like with proteins. Here, too, only relatively low intensities are required (a few hundred watts per square centimeter).

Generalizations

The two states A and B must be distinguishable, but fluorescence does not necessarily have to be involved. Switching between an absorbing and a non-absorbing state or a scattering and a non-scattering state would also be possible.

swell

  • Stefan W. Hell: Microscopy and its focal switch . In: Nature Methods . Vol. 6, No. 1 , 2009, p. 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 .

Web links

Individual evidence

  1. a b Stefan W. Hell, Jan Wichmann: Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy . In: Optics Letters . tape 19 , no. 11 , 1994, pp. 780-782 , doi : 10.1364 / OL.19.000780 .
  2. a b Thomas A. Klar, Stefan W. Hell: Subdiffraction resolution in far-field fluorescence microscopy . In: Optics Letters . Vol. 24, No. 14 , 1999, p. 954-956 , doi : 10.1364 / OL.24.000954 .
  3. a b The method was developed in 1994, theoretically described in 1995 and experimentally demonstrated in 1997:
    • Volker Dose: Peer review . In: EPL, A Letters Journal Exploring the Frontiers of Physics . Vol. 89, 2009, doi : 10.1209 / 0295-5075 / 86/10000 .
    • Stefan W. Hell, M. Kroug: Ground-state-depletion fluorescence microscopy: a concept for breaking the diffraction resolution limit . In: Applied Physics B: Lasers and Optics . Vol. 60, No. 5 , 1995, p. 495-497 , doi : 10.1007 / BF01081333 .
    • Stefan Bretschneider, Christian Eggeling, Stefan W. Hell: Breaking the diffraction barrier in fluorescence microscopy by optical shelving . In: Physical Review Letters . Vol. 98, No. 5 , 2007, p. 218103 , doi : 10.1103 / PhysRevLett.98.218103 .
  4. ^ Eva Rittweger, Dominik Wildanger, Stefan W. Hell: Far-field fluorescence nanoscopy of diamond color centers by ground state depletion . In: EPL, A Letters Journal Exploring the Frontiers of Physics . tape 86 , 2009, pp. 14001 , doi : 10.1209 / 0295-5075 / 86/14001 .
  5. Rainer Heintzmann, Thomas M. Jovin, Christoph Cremer: Saturated patterned excitation microscopy concept for optical resolution improvement . In: Journal of the Optical Society of America A . Vol. 19, No. 8 , 2002, p. 1599-1609 , doi : 10.1364 / JOSAA.19.001599 .
  6. ^ Mats GL Gustafsson: Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution . In: Proceedings of the National Academy of Sciences . Vol. 102, No. 37 , 2005, pp. 13081-13086 , doi : 10.1073 / pnas.0406877102 .
  7. Michael Hofmann, Christian Eggeling, Stefan Jakobs, Stefan W. Hell: Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins . In: Proceedings of the National Academy of Sciences . Vol. 102, No. 49 , 2005, pp. 17565-17569 , doi : 10.1073 / pnas.0506010102 .
  8. Ulrike Böhm, Stefan W. Hell, Roman Schmidt: 4Pi-RESOLFT nanoscopy . In: Nature Communications . Vol. 7, No. 10504 , 2016, p. 1-8 , doi : 10.1038 / ncomms10504 .
  9. Mariano Bossi, Jonas Fölling, Marcus Dyba, Volker Westphal, Stefan W. Hell: Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability . In: New Journal of Physics . Vol. 8, 2006, pp. 275 , doi : 10.1088 / 1367-2630 / 8/11/275 .
  10. Stefan W. Hell: Strategy for far-field optical imaging and writing without diffraction limit . In: Physics Letters A . Vol. 326, No. 1–2 , 2004, ISSN  0375-9601 , pp. 140-145 , doi : 10.1016 / j.physleta.2004.03.082 .