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Microscopy Method Resolves Fluorescence at Nanometer Scale

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Researchers led by Stefan Hell at the Max Planck Institute (MPI) for Biophysical Chemistry and the Heidelberg-based MPI for Medical Research have developed a light microscopy method, MINSTED, that is able to resolve fluorescently labeled details with molecular sharpness. The technique derives from STED microscopy, also developed by Hell, for which he received the 2014 Nobel Prize in chemistry. That method was able to achieve a resolution of 20 to 30 nm, approximately 10× sharper than the light microscopes available at the time. 

“A good 20 years ago, we fundamentally broke the diffraction resolution limit of fluorescence microscopy with STED; until then that was considered impossible,” Hell said. “Back then we dreamed: With STED we want to become so good that one day we will be able to separate molecules that are only a few nanometers apart. Now we’ve succeeded.”

MINSTED also builds on a development Hell and his team achieved in 2016, called MINFLUX, which combined an element from the STED principle with one from yet another light microscopic technique, PALM/STORM. This method achieved a resolution of just a few nanometers.

Hell theorized then that MINFLUX would not remain the only molecular resolution method, but rather would represent the first member of a new family of techniques with that level of resolution. MINSTED demonstrated that supposition to be true. The method relies on the original STED principle far more than MINFLUX.

“This has advantages. Like MINFLUX, it achieves molecular resolution, but the background noise is lower,” said Ph.D. student Michael Weber of Hell’s lab and a developer of the method. “In addition, the resolution can now be adjusted almost continuously from 200 nm down to the molecular size — 1 nm.”

The new method brings STED toward its full potential. STED microscopy, which works by switching neighboring fluorescent features or molecules on and off, one after another, uses a laser beam to stimulate molecules, which is followed by a second beam. That so-called STED beam prevents the molecules from fluorescing. The STED beam, however, is hollow — a cross section of the beam would show a holed spherical shape. Only the molecules in the middle of this doughnut-shaped beam can fluoresce. Therefore one always knows the location of the emitting molecules. 

In practice, STED isn’t able to achieve molecular resolution because the doughnut-shaped fluorescence inhibition beam can’t be made strong enough to the point where only a single molecule is able to fit into the hole.

To resolve that issue, the fluorescent molecules in MINSTED are initially isolated by randomly switching them on through an independent photochemical switching process, rather than by the doughnut beam itself. The fluorescence-preventing STED doughnut beam individually locates the fluorescent molecules, with the hole serving as a reference point

“If the hole coincides with the molecule, the molecule glows most strongly and you can figure out precisely where it is because the exact position of the STED doughnut beam is always known,” said Marcel Leutenegger, a postdoctoral researcher in Hell’s department. “That is why we gradually approach the molecules with an accuracy of one to three nanometers, that is, the size of the molecules. In connection with the photochemical on-and-off switching, the resolution becomes molecular scale.”

The research was published in Nature Photonics (www.doi.org/10.1038/s41566-021-00774-2).

BioPhotonics
May/Jun 2021
GLOSSARY
fluorescence
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
fluorescence microscopy
Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction.
Research & TechnologyMicroscopysuperresolutionSTEDSTED microscopySTED beamsMINFLUXMINSTEDPALMSTORMlasersfluorescencefluorescence microscopymolecular resolutionStefan HellMax Planck InstituteMax Planck Institut für Biophysikalische ChemieMax Planck Institute for Biophysical ChemistryBioScan

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