Nanometre imaging with fluorescence microscope


Rebecca Pool

Tuesday, January 17, 2017 - 11:45
Image: Stefan Hell and colleagues with their latest microscope, MINFLUX.
Researchers, including Nobel laureate Professor Stefan Hell, at the Max Planck Institute for Biophysical Chemistry have developed a new fluorescence microscope that can, for the first time, optically separate molecules that are only nanometres apart.
So-called MINFLUX is said to surpass even the best super-resolution light microscopy methods to date, by up to 20 times.
Key methods include STED developed by Hell and PALM/STORM described by Nobel laureate Eric Betzig, which achieve a separation sharpness of about 20 to 30 nm.
With MINFLUX, users can follow faster movements than is possible with STED or PALM/STORM microscopy. Left: Movement pattern of 30S ribosomes (parts of protein factories, coloured) in an E. coli bacterium (black-white). Right: Movement pattern of a single 30S ribosome (green) shown enlarged. [MPI f. Biophysical Chemistry/ Y. Eilers]
For MINFLUX, Hell combined the advantages of STED and PALM/STORM to open up new opportunities for researchers to investigate how life functions at the molecular level.
"We have routinely achieved resolutions of a nanometre with MINFLUX; the ultimate limit of what is possible in fluorescence microscopy," explains Hell.
"I am convinced that MINFLUX microscopes have the potential to become one of the most fundamental tools of cell biology," he adds. "With this concept it will be possible to map cells in molecular detail and to observe the rapid processes in their interior in real time."
Fluorescing molecules: MINFLUX microscopy can, for the first time, separate molecules optically which are only a few nanometres apart. PALM/STORM microscopy (right) delivers a diffuse image of the molecules while the position of the individual molecules can be easily discerned with MINFLUX (middle). [Klaus Gwosch / Max Planck Institute for Biophysical Chemistry]
Both STED and PALM/STORM separate neighbouring fluorescing molecules by switching them on and off, one after the other, so that the molecules fluoresce sequentially.
However, the methods differ in one essential point: STED microscopy uses a doughnut-shaped laser beam to turn off molecular fluorescence at a fixed location in the sample.
While this laser defines exactly at which point in space the corresponding glowing molecule is located, in practice, the laser beam is not strong enough to confine the emission to a single molecule at the doughnut centre.
In contrast, for PALM/STORM, the switching on and off is at random locations and at the single-molecule level.
This means that although the user is working at the single-molecule level, he or she does not know exact molecule positions in space; more than 50,000 detected fluorescence photons are needed to attain a resolution of less than 10 nm.
To overcome these issues, Hell combined the strengths of both methods to create MINFLUX; MINimal emission FLUXes.
Like PALM/STORM, this switches individual molecules randomly on and off but at the same time, the exact positions are determined with a doughnut-shaped laser beam, STED.
Crucially, however, the doughnut beam excites the fluorescence.
This means that if the molecule is on the ring, it will glow, and if it is exactly at the dark centre, it will not glow. In this way, the user can locate the molecule's position.
According to Hell, his colleague, Francisco Balzarotti, developed an algorithm that exploits the potential of the doughnut excitation beam, so molecule position can be quickly located with high precision.
Ribosomes in motion: Movement patterns of two different 30S ribosomes (components of protein factories, blue and orange) in an E. coli bacterial cell (black and white). In order to visualize the extremely fast movements of the 30S ribosomes, the video was slowed down 50x.
"MINFLUX is much faster in [than STED and PALM/STORM]," adds Hell. "Since it works with a doughnut laser beam, it requires much lower light signal, that is, fewer fluorescence photons per molecule as compared to PALM/STORM for attaining the ultimate resolution."
Hell and colleagues have already filmed the movement of molecules in a living E. coli bacterium with MINFLUX with unprecedented spatio-temporal resolution.
"Tracking single fluorescent proteins by MINFLUX increased the temporal resolution and the number of localizations per trace by 100-fold, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli," highlights Hell.
The researchers now intend to investigate even faster changes in living cells, such as the movement of cellular nanomachines or the folding of proteins.
Research is published in Science.
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