In the 1990s an optical imaging technique emerged that overturned the “diffraction limit”, which for over a century had defined the maximum achievable resolution an optical microscope could achieve at around half the wavelength of the illuminating light. At King’s College London’s Wheatstone Lecture 2020, attendees heard from Stefan Hell, the Nobel laureate who had developed the technique – stimulated emission depletion microscopy (STED). In his talk he described a new kid on the block in the world of optical imaging techniques that can beat the resolution of STED by a further factor of 10.
Hell began his talk with an overview of his STED technique and two others developed in the 2000s – photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) – that together led to the award of the 2014 Nobel Prize for Chemistry to Eric Betzig, Stefan W. Hell and William E. Moerner “for the development of super-resolved fluorescence microscopy.”
“I’m still struggling with this chemistry thing,” smiled Hell “I don’t know much about chemistry.” Despite the modesty of his claim, as his talk pointed out all these super-resolution techniques hinge on the use of fluorescing molecules that stain the sample being imaged. The chemistry becomes important for choosing the right molecules that will fluoresce with the right behaviour – emitting photons and “bleaching” or ceasing to emit them when flooded with light, only in ways that allow the techniques to work. Nonetheless he was true to his word that there wouldn’t be too much chemistry in the talk, giving instead a tour de force of the physics behind these techniques.
Both STED and PALM/STORM illuminate a diffraction-limited region to excite the molecules there to fluoresce. However, STED uses an additional, for example, doughnut-shaped beam to deplete emissions from part of this region, while PALM and STORM use the stochastic nature of the molecules’ fluorescence and bleaching to build up a picture with a resolution that beats the diffraction limit. Both techniques should be capable of resolution at the molecular level but as Hell pointed out, in practice they are limited to a resolution of ten times this at around 20 nm. This is still ten times better than diffraction-limited optical microscopy can achieve, but he was keen to resolve molecular level detail, which is what is achieved by his group’s new technique “MINFLUX”.
Hell described the Achilles heel of the previous super-resolution techniques – the sheer number of photons needed. In contrast MINFLUX operates by the absence of photons emitted. It tracks fluorescing molecules with a doughnut shaped illuminating beam where fluorescence is suppressed in the centre, and uses the known mathematical description of how that fluorescence changes from the centre of the hole in the beam to pinpoint the molecule from what photons have been emitted. In an ideal world tracking the molecule with it dead centre of the beam would involve no fluorescence at all. This not only liberates the achieved resolution from a performance limited by the number of photons involved but means that images can be collected much faster – something biologists love. He showed a movie of a protein moving around in an e coli cell where the technique tracked 8000 protein localizations a second.
The Wheatstone Lectures are an annual event at King’s College London that attract lay public and academics alike. The excitement of one University College London student pursuing a Masters on super-resolution techniques was infectious as he waited to hear the man who had won a Nobel Prize for work in this field describe the state of the art. The lectures commemorate the life and work of one of the college’s alumni Charles Wheatstone (1802-1875), a scientist and prolific inventor whose legacy includes the symphonium, the stereoscope and work on establishing the telegraph system and the Wheatstone Bridge.
Photo credit: King’s College London Department of Physics
Words: Dr Anna Demming