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Microscopy beyond the limit of resolution

The Polish-Israeli team led by Dr. Radek Łapkiewicz from the Faculty of Physics at the University of Warsaw presented a new, revolutionary method of microscopy that theoretically has no resolution limit in the magazine "Optica".

The research was announced by the Foundation for Polish Science (FNP) in a communication to PAP. Dr. Łapkiewicz is a recipient of the FIRST TEAM program.

The development of life sciences and medicine requires the observation of ever smaller objects - for example the structure and interaction of proteins in cells. The samples observed should not differ from the structures naturally occurring in the body - therefore the methods and reagents must not be used too aggressively.
The classic optical microscope has insufficient resolution. Due to the wavelength of the light, such a microscope does not allow the imaging of structures that are smaller than about 250 nanometers (half the wavelength of green light). Objects that are closer together can no longer be distinguished. This is the so-called diffractive limitation.
The electron microscope has a resolution several orders of magnitude higher than a light microscope, but it allows us to only observe dead objects that are placed in a vacuum and bombarded with an electron beam. It is not about studying living organisms or processes naturally occurring in them.

Image source: Optica Vol. 7, Issue 10, pp. 1308-1316 (2020) •

The solution is a high-resolution fluorescence microscope (research into developing this area of ​​imaging won the Nobel Prize in 2008 and 2014). As recalled in the communication, several fluorescence microscopy techniques already exist. PALM, STORM or STED methods are characterized by a high resolution and allow the differentiation of objects that are only a dozen nanometers apart. However, a long exposure time and a complicated preparation of the biological preparations are required. SIM or ISM microscopy, on the other hand, are methods that are easy to use, but with significantly limited resolution - they allow structures to be seen that are only twice as small as the diffraction limit. Dr. Radek Łapkiewicz from the Quantum Optics Laboratory at the Faculty of Physics at the University of Warsaw and Aleksandra Środa and Adrian Makowski - students from the Warsaw University of Life Sciences - together with Dan Oron's team from the Weizmann Institute in Israel, have improved the existing ISM method and introduced a new technology the super-resolution optical fluctuation image scanning microscopy (SOFISM). They managed to show that the diffraction limit was overcome four times, reported the FNP.

"SOFISM offers a compromise between ease of use and resolution. We believe that our method can fill the niche between complex, difficult-to-use, very high-resolution techniques and low-resolution but easy-to-use methods. SOFISM has no theoretical limit of resolution, but it does have in our work we presented results in which we exceeded the diffraction limit four times. In the article we also showed that the SOFISM method has a high potential for imaging three-dimensional biological structures, "says Dr. Radek Łapkiewicz, who is quoted in the communication.

The method developed by Warsaw physicists is technically very accessible. It is sufficient - as we read in the communiqué - to slightly modify the confocal microscope commonly used in laboratories (replacement of the photomultiplier with a SPAD array detector), to slightly extend the measurement time and to change the data handling procedure.

"Until recently, SPAD array detectors were expensive and inadequate for applications like ours. That situation has changed recently. New SPAD detectors have been available since last year that incorporate both technology and the price barriers have been removed. Therefore, we believe that fluorescence microscopic techniques such as SOFISM can become common techniques for microscopic examinations within a few years ". - emphasizes Dr. Łapkiewicz.

The publication is here to find.