Researchers from the Faculty of Physics of the University of Warsaw and the Weizmann Institute of Science have developed a new method of fluorescent microscopy which, in theory, has no resolution limit. Their research is published in the Optica journal.
Optical microscopes does not allow imaging structures smaller than roughly 250 nanometers. As a result, objects closer than 250 nm (for green light) are difficult to discern. This phenomenon, known as the diffraction limit, is one of the primary obstacles in observing small biological structures. Electron microscopes provide orders of magnitude better resolution but are limited to the examination of inanimate objects, subjected to an electron beam. The development of super-resolution fluorescence microscopy is the best solution to high magnitude resolution of animate biological objects.
Several techniques of fluorescence microscopy are available, including PALM, STORM or STED microscopy, which are characterized by an ultra-high resolution and allow discerning objects located just a dozen or so nanometers from each other. However, these techniques are limited by long exposure times and a complex procedure of biological specimen preparation. Other techniques, such as SIM or ISM microscopy, are easier to work with, but offer a very limited resolution improvement.
Aleksandra Sroda, Adrian Makowski and Dr. Radek Lapkiewicz from the Quantum Optics Lab at the Faculty of Physics of the University of Warsaw, in cooperation with Prof. Dan Oron's team from the Weizmann Institute of Science in Israel, have developed a new technique of super-resolution microscopy, called Super-resolution optical fluctuation image scanning microscopy (SOFISM). In SOFISM, the naturally occurring fluctuations in emission intensity of fluorescent markers are utilized to enhance the spatial resolution of an image scanning microscope (ISM).
"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 easy-to-use lower-resolution methods. SOFISM does not have a theoretical resolution limit, and in our article, we demonstrate results that 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 Dr. Radek Lapkiewicz.
He continues, "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".