They can make tiny cell structures visible: Modern light microscopes offer resolutions of a few tenths of a nanometer, or one millionth of a millimeter. So far, high-resolution microscopes were much slower than conventional methods, because more or finer image data had to be recorded. Researchers at the University of Bielefeld have now further developed the high-resolution SR-SIM process together with partners from Jena. The scientists show that SR-SIM is also possible in real time and with a very high imaging rate ̵
"That's why this type of microscopy is very useful for biology or medicine applications, so far the problem is that microscopes with a sufficiently high resolution can not display information at the proper speed," says Professor Dr. med. Thomas Huser heads the Biomolecular Physics group at the University of Bielefeld. The SR-SIM project is funded by the German Research Foundation (DFG) and the European Union in the framework of Marie Skłodowska-Curie actions.
SR-SIM stands for "Super Resolution Structured Illumination Microscopy" and is a fluorescence microscopy method. Objects are irradiated with laser light. This light excites specific fluorescent molecules in the sample to re-emit light of a different wavelength. The microscopic image then shows the re-emitted light. "In contrast to other conventional fluorescence microscopy methods, SR-SIM does not illuminate the samples evenly, but with a fine, lattice-like pattern, which enables a much higher resolution," says Huser.
The procedure consists of two steps: The light emitted by the sample is first recorded in several individual images. The finished image is then reconstructed on a computer from this raw data. "Especially the second step has cost a lot of time so far," says Andreas Markwirth, also a member of the working group Biomolecular Physics of the University of Bielefeld and lead author of the study. The Bielefeld researchers therefore worked with Professor Dr. Ing. Rainer Heintzmann from the Leibniz Institute for Photonic Technologies and the Friedrich Schiller University in Jena to accelerate the process. The microscope is now designed to generate raw data faster. In addition, thanks to parallel computer processing on modern graphics cards, image reconstruction takes considerably less time.
For their study, the researchers tested the new method on biological cells and recorded the motions of mitochondria, cell organelles, about 1 micron in size. "We were able to produce about 60 frames per second – a higher frame rate than cinematographic films – the time between measurement and image is less than 250 milliseconds, so the technology allows real-time imaging," says Markwirth.
Up So far, super-resolution methods have often been combined with conventional methods: Structures are first discovered using a conventional fast microscope. These structures can then be examined in detail with a high-resolution microscope. "However, some structures are so small that they can not be found with conventional microscopes, such as certain pores in liver cells, and our method is both high-resolution and fast, allowing biologists to study such structures," says Huser. Another application for the new microscope is the study of virus particles on their way through the cell. "So we can understand exactly what happens in infection processes," says Huser. He expects the microscope to be used for such studies at the University of Bielefeld next year.
High-resolution microscopes have only been around for about 20 years. In 1873, Ernst Abbe discovered that the resolution of a visible light optical system is limited to about 250 nanometers. In recent years, however, several optical methods have been developed to break the limit known as Abbe's diffraction barrier. William E. Moerner and Eric Betzig from the USA and Stefan Hell from Germany were awarded the Nobel Prize for Chemistry in 2014 for the development of a super-resolution in the range of approximately 20 to 30 nanometers.
New open-source software for high-resolution microscopy
Andreas Markwirth et al. Multicolor Structured Illumination Microscopy with Video Rate and Simultaneous Real-Time Reconstruction, Nature Communications (2019). DOI: 10.1038 / s41467-019-12165-x
HD Microscopy in Milliseconds (September 20, 2019)
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