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MINFLUX enhances super-resolution microscopy

13 Jun 2024

EMBL project simplifies protocol for imaging of cells and tracking fluorophores.

MINFLUX is a super-resolution microscopy protocol designed to improve the localizing of specific photon emitters in space.

Developed in 2016 by a German team including super-resolution pioneer and Nobel prize laureate Stefan Hell, MINFLUX involves probing an emitter with a local minimum of excitation light - ie. "minimal photon flux" - as part of an effort to reduce the fluorescence photons needed for final precision location of the same emitter.

"As in photoactivated localization microscopy and optical reconstruction microscopy, fluorophores are switched on and off; but the emitter is located using an excitation beam that is doughnut-shaped, as in stimulated emission depletion," commented the developers in their original Science paper.

"Finding the point where emission is minimal reduces the number of photons needed to localize an emitter. MINFLUX attained 1-nanometer precision, and, in single-particle tracking, achieved a 100-fold enhancement in temporal resolution."

Variants of the MINFLUX approach have since included the pMINFLUX multiplexing technique employed at LMU Munich to determine the position of several dyes at the same time, and investigate fast dynamic processes between several molecules.

However, current implementations of MINFLUX are complicated, expensive and limited in speed and robustness, according to a project at the Heidelberg site of the European Molecular Biology Laboratory (EMBL).

"MINFLUX setups are complex and require specialized equipment, which has limited the widespread use of this powerful technique," noted EMBL. "Traditional methods often involve bulky and expensive components, making MINFLUX inaccessible to many research labs."

As described in Light Science & Applications EMBL set out to improve this situation with a novel MINFLUX excitation module based on a novel variable phase plate, enabling 3D multi-color excitation at high resolution.

Simpler set-up unlocks new knowledge

The new design combines two simpler devices: a spatial light modulator and an electro-optical modulator, with the former manipulating the patterns of light used for excitation of fluorophores and the latter controlling the intensity of the light. This combination is significantly faster, cheaper, and easier to use than traditional methods, according to EMBL.

"Using simpler components allows for much faster scanning of the light pattern, improving the accuracy of measurements for sharper and more detailed images of the molecules," commented the EMBL developers.

"Secondly, the more straightforward setup significantly reduces the cost of the equipment needed for MINFLUX, making this powerful technique more accessible to researchers. Finally, the new method is more user-friendly and can be more easily integrated into existing microscopes, streamlining the research process."

As proof-of-concept EMBL tested its module in a low-NA set-up, rather than a full MINFLUX microscope, and modelled a range of super-resolution illumination patterns. It found that excitation light could be scanned across a fluorophore within a microsecond, switched within 60 microseconds, and alternated among different excitation wavelengths.

Expanding this approach should enable further evolutions of the process, and EMBL envisages multi-color MINFLUX microscopy with the localization of two or more fluorophores from the same module.

"This could open up new possibilities for studying a wide range of biological processes at the molecular level," said EMBL. "With MINFLUX becoming more accessible, scientists can delve deeper into the unseen world, unlocking new knowledge about how life works at its most fundamental level."

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