Microscopy Beyond the Resolution Limit

The team from the Faculty of Physics of the University of Warsaw and the Weizmann Institute of Science in Rehovot, Israel, has made another signi­ficant achieve­ment in fluorescent micro­scopy. The team presented a new method of micro­scopy which, in theory, has no resolution limit. In practice, the team managed to demonstrate a fourfold improve­ment over the diffraction limit.

Image of microtubules in a fixed cell sample. A 3 μm × 3 μm confocal scan of microtubules in a fixed 3T3 cell labelled 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. (Source: A. Makowski, UW Physics)

The continued development of biological sciences and medicine requires the ability to examine smaller and smaller objects. Nowadays several techniques of fluores­cence micro­scopy are available, 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 improvement, allowing to identify structures only half the size of the dif­fraction limit.

Aleksandra Sroda, Adrian Makowski and Radek Lapkiewicz from the Quantum Optics Lab at the Faculty of Physics of the University of Warsaw, in cooperation with Dan Oron’s team from the Weizmann Institute of Science in Israel, have introduced a new technique of super-reso­lution micro­scopy – Super-resolution optical fluc­tuation image scanning microscopy SOFISM. In SOFISM, the naturally occurring fluc­tuations in emission intensity of fluorescent markers are used to further enhance the spatial resolution of an image scanning microscope (ISM). ISM, an emerging super-reso­lution method, has already been implemented in commercial products and proven valuable for the bio-imaging community.

Largely, since it achieves a modest improve­ment in lateral resolution (x2), with very few changes to the optical setup and without the common handicap of long exposure times. Thus, it enables a natural extension of the capa­bilities of a standard confocal micro­scope. ISM uses a confocal microscope in which a single detector is replaced with a detector array. In SOFISM corre­lations of intensities detected by multiple detectors are computed. In principle, the measure­ment of the n-th order correlation can lead to a factor of 2n resolution improvement with respect to the diffrac­tion limit. In practice, the resolution achievable for higher-order corre­lations is limited by the signal-to-noise ratio of the measure­ments.

“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 a high potential in the imaging of three-dimen­sional bio­logical structures,” said Radek Lapkiewicz.

Crucially, SOFISM 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 Lapkiewicz. (Source: U. Warsaw)

Reference: A. Sroda et al.: SOFISM: Super-resolution optical fluctuation image scanning microscopy, Optica 7, 1308 (2020); DOI: 10.1364/OPTICA.399600

Link: Quantum Optics Lab, Faculty of Physics, University of Warsaw, Warsaw, Poland

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