Microscopy beyond the resolution limit

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The Polish-Israeli team led by Radek Łapkiewicz from the Faculty of Physics of the University of Warsaw, winner of the FIRST TEAM programme of the Foundation for Polish Science, has made another significant achievement. In the pages of the Optica journal, the scientists presented a new method of microscopy, which, in theory, has no resolution limit.  In practice, the team managed to demonstrate a fourfold improvement over what is known as the diffraction limit, which results from the nature of light, and is one of the main obstacles in observing the tiniest biological structures.

The continued development of biological sciences and medicine requires the ability to examine smaller and smaller objects. Scientists need to see into the structure of, and the mutual relationships between, for example, proteins in cells. At the same time, the samples being observed should not differ from the structures naturally occurring in the body, which rules out the use of aggressive procedures and reagents. Although it revolutionised the natural sciences, the classic optical microscope is clearly insufficient today. Due to the wavelike nature of light, an optical microscope does not allow imaging structures smaller than about 250 nanometres. As a result, objects closer to each other than half the wavelength of light (which is about 250 nm for green light) cannot be discerned. It is this phenomenon, known as the diffraction limit, that scientists have long attempted to overcome. Electron microscopes provide orders of magnitude better resolution, but only allow the examination of inanimate objects, which must be placed in a vacuum and bombarded by an electron beam. For this reason, electron microscopy cannot be used for studying living organisms and the natural processes occurring in them. This is where fluorescence microscopy steps in, hence the rapid development of super-resolution fluorescence microscopy as a field of physical sciences and the two Nobel Prizes already awarded for related research ? in 2008 and 2014.

Nowadays several techniques of fluorescence microscopy are already here, and some of them have become widespread in biological imaging. Some methods, such as PALM, STORM or STED microscopy, are characterised by an ultra-high resolution and allow discerning objects located just a dozen or so nanometres from each other. However, these techniques require long exposure times and a complex procedure of biological specimen preparation. Other techniques, such as SIM or ISM microscopy, are easy to use, but offer a very limited resolution, allowing to identify structures only half the size of the diffraction limit.

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Dr Radek Łapkiewicz from the Quantum Optics Lab at the Faculty of Physics of the University of Warsaw (FP UW), and Aleksandra Środa and Adrian Makowski ? FP UW students, in cooperation with Prof. Dan Oron?s team from the Weizmann Institute of Science in Israel, have introduceda new technique of super-resolution microscopy, called Super-resolution optical fluctuation image scanning microscopy (SOFISM). ?SOFISM is a compromise between ease of use and resolution. We believe that our method will fill the niche between the complex, difficult-to-use techniques providing very high resolution and the easy-to-use lower-resolution methods. SOFISM does not have a theoretical resolution limit, and in our article we demonstrate results which are four times better than the diffraction limit. We also show that the SOFISM method has huge potential in the imaging of three-dimensional biological structures,? said dr Radek Łapkiewicz.

Crucially, the method developed by Warsaw-based physicists is, in its technical aspects, highly accessible, as it only requires introducing a small modification to the widely-used confocal microscope ? replacing its photomultiplier tube with a SPAD array detector. In addition, it is necessary to slightly increase the measurement time and change the data processing procedure. ?Until recently, SPAD array detectors were expensive and their specifications were not sufficient for correlation based microscopy. This situation has recently changed. The new SPAD detectors introduced last year removed both the technological and price-related barriers. This makes us think that fluorescence microscopy techniques such as SOFISM might, in a few years? time, become widely used in the field of microscopic examination,? stressed dr Łapkiewicz.

Source: Aleksandra Środa, Adrian Makowski, Ron Tenne, Uri Rossman, Gur Lubin, Dan Oron, Radek Łapkiewicz: ?SOFISM: Super-resolution optical ?uctuation image scanning microscopy?.

More information:

Figure description: Image of microtubules in a ?xed cell sample. A 3 ?m × 3 ?m confocal scan of microtubules in a ?xed 3T3 cell labeled with quantum dots analyzed in two ways. Upper left: image scanning microscopy (ISM), lower right: super-resolution optical fluctuation image scanning microscopy (SOFISM) after fourier-reweighting.

The FIRST TEAM Programme is implemented by the Foundation from EU Funds within the European Regional Development Fund as part of the Smart Growth Operational Programme, Axis IV: Increasing the research potential, Measure 4.4. Increasing the human potential in the R&D sector.