A team of scientists have made use of technology developed by Nobel Prize winner Stefan Hell and created a super-resolution microscope that will probe nature like never before, reports Phys.
It is the holy grail of light microscopy: improving the resolving power of this method such that one can individually discern molecules that are very close to each other.
The new fluorescence microscope will allow researchers to discern elements that are only nanometers (one millionth of a millimeter) apart from each other.
The super-resolution microscope MINFLUX was developed by a team at the Max Planck Institute for Biophysical Chemistry in Göttingen.
MINFLUX surpasses conventional light microscopes by 100 times.
The most effective super-resolution microscopes previously developed were PALM/STORM, described by Nobel laureate Eric Betzig, and a STED microscope, developed by Hell himself.
MINFLUX surpasses even those models by 20 times. It makes use of the technologies underpinning the STED and PALM/Storm devices.
STED is an acronym for “Stimulated emission depletion microscopy.” The technology was developed by Stefan Hell, in collaboration with Jan Wichmann, in 1994.
STED is one of the techniques that make up super-resolution microscopy. It works by selectively deactivating the fluorophores active during imaging, thereby minimizing the area of illumination at the focal point. This allows super-resolution images to be created.
In other words, STED interrupts the normal process of fluorescence. Normal fluorescence occurs when an electron is excited from the ground state, releasing a photon. STED interrupts this process before the photon is released
It was the STED technology that won the Nobel Prize in Chemistry for Hell in 2014.
MINFLUX develops on this previous breakthrough even further. Hell told journalists, “We have routinely achieved resolutions of a nanometer with MINFLUX, which is the diameter of individual molecules – the ultimate limit of what is possible in fluorescence microscope”
PALM/Storm has also been surpassed by MINFLUX.
PALM stands for ‘Photoactivated localization microscopy” and Storm stands for “stochastic optical reconstruction microscopy.” In these techniques, a fluorescence microscope is used to collect a large number of images, each containing just a few active isolated fluorophores. Each fluorophore will have been activated from a non-emissive to an emissive state throughout the course of the image series, which gives researchers as much information as possible about the molecules.
Consider two molecules very close together. If photon emissions from two neighboring fluorescent molecules can be made distinguishable, i.e. the photons coming from each of the two can be identified and “separated,” then it is possible to create super-resolution images because we have a way of distinguishing them.
In PALM/Storm, only a fraction of fluorophores are “switched on,” therefore only a fraction are optically resolvable from the rest at a given moment. Thousands of such on-off cycles are incorporated through the taking of a series of images. This information is combined and used to reconstruct a super-resolution image.
MINFLUX makes use of both these approaches, incorporating the advantages of both STED and PALM/Storm and combining them into one powerful piece of technology.
Hell told reporters, “I am convinced that MINFLUX microscopes have the potential to become one of the most fundamental tools of cell biology. 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. This could revolutionize our knowledge of the molecular processes occurring in living cells.”
MINFLUX uses a laser to manipulate the photon emission process in selected particles, like STED, but it also uses the activator/reporter pair model in PALM/Storm, switching neighboring molecules on and off.
Ian Smith of the University of CA describes the similarities between the technologies this way:
All approaches are based on the same principle – localizing individual molecules one at a time, by ensuring that only sparsely distributed molecules are fluorescing at a given time.
It’s worth noting that, unlike STED, MINFLUX uses a laser to excite certain molecules to fluorescence, rather than interrupting the fluorescence process.
MINFLUX, like PALM/STORM, switches individual molecules randomly on and off. However, at the same time, their exact positions are determined with a doughnut-shaped laser beam as in STED.
Nobel prize winner Hell told reporters that MINFLUX is also much faster than the previous technologies. “MINFLUX is much faster in comparison. Since it works with a doughnut laser beam, it requires much lower light signal, i.e. fewer fluorescence photons, per molecule as compared to PALM/STORM for attaining the ultimate resolution.”
The team demonstrated the technique by filming the movement of two different 30S ribosomes responsible for protein synthesis in a living E coli bacteria. The scientists were able to resolve arrays of single fluorescent molecules at 6nm intervals, reports Chemistry World.
Researcher Steven Lee said, ‘These are exciting times for super-resolution imaging in general and we hope to see how these technical tools will be transferred and adopted by the biological community to address the most important questions in biomedicine.’
[Featured Image by Christopher Furlong/Getty Images]