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