Novel microscopy in the extreme ultraviolet reveals the nanoworld of microorganisms

Interdisciplinary cooperation at the Beutenberg honored with Beutenberg Campus Award

Jena, Germany  | November 16, 2023

An interdisciplinary team of researchers has developed a novel, high-resolution and lensless microscope that works with extreme ultraviolet (EUV) light. The microscope enables imaging of biological samples with a remarkable resolution of up to 16 nanometers. The interdisciplinary collaboration, in which several partners from the Jena Beutenberg Campus were involved, has now been awarded the Beutenberg Campus Jena e.V. Science Prize.

In order to develop more targeted treatment methods for diseases, precise knowledge of the different stages of pathogen development and their interaction with infected cells is required. Extreme ultraviolet (EUV) light with a very short wavelength is particularly suitable for this, as it allows microscopy with a very high resolution (less than 20 nanometers).

© Beutenberg-Campus Jena e.V., Tina Peißker
The first winners of the Life and Physical Sciences Award in the newly introduced category "Excellent Interdisciplinary Cooperation" are Dr. Jan Rothhardt (FSU-IAP; 2nd from left), Prof. Dr. Thomas Pertsch (FSU-IAP, center) and Prof. Dr. Jens Limpert (FSU-IAP, Fraunhofer IOF; left in the picture) as well as Prof. Dr. Falk Hillmann (formerly Leibniz-HKI), who unfortunately could not accept the award in person. The laudatory speech was given by Dr. Ramona Eberhardt (2nd from right) from Fraunhofer IOF, before the chairman of the association, Prof. Peter Zipfel (right in the picture), presented the award.

EUV enables high spatial resolution for the smallest cell structures


An interdisciplinary research team led by Dr. Jan Rothhardt - a member of Fraunhofer IOF and the Institute of Applied Physics (IAP) at the Friedrich Schiller University Jena and the Helmholtz Institute Jena - has now been able to examine complex microbiological samples, specifically seedlings of the fungus Aspergillus nidulans and Escherichia coli bacteria, using a new type of high-resolution lensless microscope that works with extreme ultraviolet (EUV) light. This made it possible, for example, to identify the Spitzenkörper of A. nidulans and the chemical composition of the cell wall of E. coli bacteria at different stages of cell division. [1]

"The high elemental contrast of the EUV radiation and an exceptionally high spatial resolution make it possible to identify subcellular structures of the two model organisms and subsequently assign them to known organelles," says Dr. Falk Hillmann, researcher for the Evolution of Microbial Interactions at the Leibniz Hans Knöll Institute in Jena, impressed. His team selected the biological model samples and developed a suitable preparation method on EUV-transparent silicon nitrite membranes.

Functionality of the new EUV microscope


But how exactly does this new type of imaging work and what technical innovations did it require? "A novel EUV microscope adapted to the source was realized in my working group. It is based on the ptychography method, a special form of computer-assisted, lensless imaging. [2] The microscope is the first to use structured EUV illumination, which is crucial for high resolution and excellent image quality. [3] 'Structured' in this context means that the EUV radiation used to illuminate the sample is shaped by a special illumination mask. This mask is designed in such a way that it transmits certain structures in the nanometer range to the EUV radiation. These can be patterns, lines or other shapes, which are then projected onto the biological sample. The structured EUV exposure plays a crucial role in the microscope's high resolution and excellent image quality," explains Dr. Rothhardt.

The illumination mask is essential for the resolution and image quality of the microscopic image. Prof. Thomas Pertsch, Professor of Nano and Quantum Optics, IAP at the Friedrich Schiller University Jena, who has driven its development, emphasizes: "The production of this EUV mask requires the highest precision. A process has been developed with which absorbing binary EUV masks can be produced on the basis of thin metal foils. The nanostructuring was applied to the masks using a focused ion beam. By combining these nanostructured masks, the unique EUV light source and the novel EUV microscope, we were able to achieve a record resolution of 16 nanometers and demonstrate this in a resolution test. [3] Since the EUV microscope provides quantitative amplitude and phase information in each image pixel, it is even possible to identify the chemical composition on the nanoscale."

High-power EUV light source developed under the leadership of the IAP and Fraunhofer IOF


The compact EUV light source required for this was developed under the leadership of Prof. Jens Limpert's working group at the IAP and the Fraunhofer IOF. Laser-driven coherent EUV light sources on a laboratory scale have undergone rapid development in recent years. Record performances have already been demonstrated at the Institute of Applied Physics, which are comparable to synchrotron beam sources in some areas of EUV [4]. However, the laser-driven EUV light sources from Jena can be operated more cost-effectively and are compact and portable. Both are decisive application advantages over large-scale synchrotron research facilities.

"The new method in the extreme ultraviolet range enables high-resolution imaging of biological samples in a previously unattainable way," says Prof. Limpert, emphasizing the importance of this scientific milestone: "The interdisciplinary collaboration has enabled us to create a compact EUV microscope on a laboratory scale and demonstrate it on microorganisms. This not only expands the possible applications, but also makes the technology more accessible."

Honored with the Science Award of the Beutenberg Campus Jena e.V.


This special interdisciplinary cooperation across the top research institutes at Beutenberg was honored on November 15 with the Science Award of the Beutenberg Campus Jena e.V., which was awarded for the first time in the category "Excellent Interdisciplinary Cooperation". "This award highlights exactly what the campus lives for and makes it special: cutting-edge research with outstanding application relevance beyond subject boundaries, in other words "...where life science meets physics!", says Dr. Christiane Meyer, Scientific Officer of the Beutenberg-Campus e.V., with delight.

Scientific publications


[1] C. Liu, W. Eschen, L. Loetgering, D. S. Molina, R. Klas, A. Iliou, M. Steinert, S. Herkersdorf, A. Kirsche, T. Pertsch, F. Hillmann, J. Limpert, and J. Rothhardt, »Visualizing the ultra-structure of microorganisms using table-top extreme ultraviolet imaging«, PhotoniX 2023 4:1 4(1), 1–15 (2022).

[2] P. Thibault, M. Dierolf, A. Menzel, O. Bunk, C. David, and F. Pfeiffer, »High-resolution scanning x-ray diffraction microscopy«, Science 321(5887), 379–82 (2008).

[3] W. Eschen, L. Loetgering, V. Schuster, R. Klas, A. Kirsche, L. Berthold, M. Steinert, T. Pertsch, H. Gross, M. Krause, J. Limpert, and J. Rothhardt, »Material-specific high-resolution table-top extreme ultraviolet microscopy«, Light: Science & Applications 2022 11:1 11(1), 1–10 (2022).

[4] R. Klas, A. Kirsche, M. Gebhardt, J. Buldt, H. Stark, S. Hädrich, J. Rothhardt, and J. Limpert, »Ultra-shortpulse high-average-power megahertz-repetition-rate coherent extreme-ultraviolet light source«, PhotoniX 2(1), 1–8 (2021).