Researchers at Goethe University Frankfurt have successfully combined two very advanced fluorescence microscopy techniques
Is it possible to watch at the level of single cells how fish embryos become trout, carp or salmon? Researchers at Goethe University Frankfurt have successfully combined two very advanced fluorescence microscopy techniques. The new high-resolution light microscope permits fascinating insights into a cell’s interior.
Using the “light-sheet microscopy” technology invented and developed by Professor Ernst Stelzer, it was already possible to observe organisms in a very precise and vivid way during cell differentiation. His group at Goethe University Frankfurt has now combined light sheets with a technique which so far only allowed very high spatial resolutions (<100nm) on a cell’s surface. Combining both methods makes it possible to obtain a three-dimensional insight into a cell with a high resolution.
Light-sheet-based fluorescence microscopy (LSFM) is the most recent three-dimensional fluorescence microscopy technique. In fluorescence microscopy, a fraction of a cell’s molecules is labelled with fluorescent markers, which are lit up with a beam of light. A camera records the three-dimensional distribution of the fluorescing molecules, i.e. the fluorophores. The outstanding advantage of LSFM is that even sensitive samples such as fish embryos survive observation. This is a major advancement since conventional methods, which illuminate the whole sample, expose the specimens to much more energy and destroy the cells in a very short period of time.
Ernst Stelzer, professor at the Institute of Cell Biology and Neuroscience and a principal investigator in the Cluster of Excellence “Macromolecular Complexes” of Goethe University Frankfurt, explains that LSFM does not illuminate the entire sample but only micrometre-thin light sheets. “Since we examine the biological specimens under conditions that are as natural as possible, we achieve very precise results”, says Stelzer. However, not only static images of cells but also dynamic changes in their environment or genetic mutations can be measured in direct comparisons.
Bo-Jui Chang, Victor Perez Meza and Ernst Stelzer have now improved the technique further: “We combined light-sheet fluorescence microscopy with coherent structured illumination microscopy (SIM). This allows for an extremely high resolution”, he reports. SIM is a super-resolution technique that produces several images, which are combined digitally. As a result, resolution is improved in the physical sense. The technical approach is to excite a fluorescing sample with a very specific illumination pattern. Sub-100 nm resolutions with this method are limited to surfaces but the technique has major advantages. It is fairly moderate in the excitation of the fluorescence, allows very fast imaging and can be used with all fluorescing molecules for high-resolution purposes.
“In the new microscope, which we call csiLSFM, we have developed the principle of SIM further in such a way that sub-100 nm resolutions are no longer limited to surfaces but can also be used in extensive three-dimensional objects. Here, two counterpropagating light sheets interfere at an angle of 180° so that they form the smallest possible linear interference pattern. As a result, we achieve an optimal resolution of less than 100 nanometres,” explains Ernst Stelzer. The new instrument has three objective lenses. It works via the flexible control of rotation, frequency and phase shift of the perfectly modulated light sheet.
Images of endoplasmic reticulum of yeast, a complex membrane network of tubules, vesicles and cisterns, show that the researchers can use csiLSFM to work successfully with physiologically important objects.
Publication: Chang BJ, Perez Meza VD, Stelzer EHK (2017) csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm. Proc Natl Acad Sci U S A, 114(19):4869-4874. doi: 10.1073/pnas.1609278114 (2017 May 9). Epub 2017 Apr 24.
A picture can be downloaded under: http://www.uni-frankfurt.de/66612661
Caption: Live yeast cell embedded in agarose. From left to right: conventional fluorescence, conventionally treated and with csiLSFM. The bar is 1 µm wide.
Photo: Stelzer Research Group, Goethe University Frankfurt
Further information: Prof. Dr. Ernst H. K. Stelzer, Institute of Cell Biology and Neuroscience, Buchmann Institute for Molecular Life Sciences,
Faculty of Biological Sciences, Riedberg Campus, Tel.: +49 (69) 798 42547/42545, email@example.com
State of Hesse grants funds of about € 525,000 for research against forgetting
Frankfurt. Boris Rhein, Minister for Science and the Arts, today introduced the holder of Germany’s first chair for research on the history and impact of the Holocaust, Professor Sybille Steinbacher, and welcomed her to her new post. Sybille Steinbacher took up office on 1 May as Director of the Fritz Bauer Institute. The position is linked to the new “Chair for Research on the History and Impact of the Holocaust” at Goethe University Frankfurt. The State of Hesse is sponsoring the Fritz Bauer Institute with € 375,100 this year and financing the Holocaust professorship with a further € 150,000, so that a total of € 525,100 of state funds is available for the two entities in 2017.
Boris Rhein, Minister for Science and the Arts: “I am very pleased that all the groundwork has now been completed and Professor Steinbacher can start to breathe life into her new task. The Holocaust professorship is a milestone along the path to a better understanding of National Socialist crimes and the history of their impact up to the present day. This joint appointment by Goethe University Frankfurt and the Fritz Bauer Institute will also further enhance the integration of university and institute-based research. I wish Professor Steinbacher every success in her work.”
Professor Steinbacher is a renowned expert in the field of Holocaust research as documented by many pertinent research papers. She already dealt with persecution and extermination policies in the National Socialist state and societal reactions to it in her Master’s thesis at LMU Munich. Her dissertation entitled “‘Model Town’ Auschwitz. Germanisation Policy and Murder of Jews in East Upper Silesia”, which she submitted at Ruhr University Bochum, later formed the basis for an internationally much acclaimed standard reference work on the subject, which was translated into numerous languages.
Professor Birgitta Wolff, President of Goethe University Frankfurt: “It is a great joy to us that we have succeeded in bringing Sybille Steinbacher to Goethe University. A type of Holocaust research such as we see it in Frankfurt can help not only to understand better the genocide of the Jews in the National Socialist era. It is equally important that the right lessons are drawn from it for the present and the future. Sybille Steinbacher’s research is highly topical in view, for example, of the fact that racially and ethnically motivated exclusion and discrimination seem at the moment to becoming socially acceptable again all around the world.”
Professor Sybille Steinbacher, Chair of Research on the History and Impact of the Holocaust and Director of the Fritz Bauer Institute: “The creation of this chair is a special event in the some 20-year history of the Fritz Bauer Institute. It signifies an affirmation and upgrading of its work, which is committed to a dual task: communication and research. Through this chair, research will in future be strengthened and the Institute and Goethe University more closely interlocked. I consider the fact that there is now a professorship in Germany with this special title to be an important achievement in terms of science and research policy. I will be tackling my new tasks with great vigour and creative drive.”
Sybille Steinbacher was already visiting professor at Goethe University Frankfurt for the history and impact of the Holocaust in 2010 in conjunction with the Fritz Bauer Institute, following which she was professor for Comparative Dictatorship, Violence and Genocide Studies at the University of Vienna. She has been Project Manager of the Dachau Symposium on Contemporary History since 2012. She is a member of several scientific bodies, including the international advisory board of the Richard Koebner Minerva Centre for German History at the Hebrew University of Jerusalem.
“The Fritz Bauer Institute is an education and research facility of highest international standing and its outreach goes far beyond the borders of Hesse. Above all the confrontation with the ethical and moral justification structures of the Holocaust up to the present day makes the research so unique and important. Especially in the land of the perpetrators there must be no forgetting. The linking of the new Holocaust professorship with the directorship of the Fritz Bauer Institute is thus a unique opportunity that we are using to give as great a boost as possible to the subject’s scientific reappraisal”, explained Boris Rhein, Minister for Science and the Arts.
A team around Ivan Dikic and Mike Heilemann discovers an inflammatory signaling platform which may lead to novel antibiotic therapies
FRANKFURT.With drug resistance being on the rise worldwide, bacterial infections pose one of the greatest global threats to human health. By deciphering the host-pathogen interaction on a molecular level, researchers hope to pave the way for new therapies. Studying the cell’s reaction to Salmonella, scientists from Goethe University Frankfurt have now made a critical discovery to this respect.
All bacteria have developed clever mechanisms for survival and propagation within host cells. Salmonella are a typical example: usually they hide in membrane-bound particles with only very few bacteria escaping to the cell’s interior. Those escapees are extremely dangerous as they proliferate and spread at enormous speed. To stop such an invasion, cells have developed very effective defense strategies. An interdisciplinary team around Prof. Ivan Dikic (Institute of Biochemistry II) and Prof. Mike Heilemann (Institute of Physical and Theoretical Chemistry), both from Goethe University Frankfurt, now studied such a cellular defense mechanism by visualizing protein patterns at the near-molecular level.
Protein chains relay pro-inflammatory signals
Upon bacterial invasion, cells react fast: They flag escaped bacteria with a small protein called ubiquitin, which is known to regulate numerous cellular processes. The attached flags contain chains of differently linked ubiquitin molecules, resulting in a secret code, which has so far only partially been decoded. Similar to mobile transmission towers, these ubiquitin chains relay specific signals from the surface of the bacteria into the cell.
Employing super-resolution microscopy, the Frankfurt team now succeeded with visualizing different ubiquitin chains on the bacterial surface and analyzing their molecular organization in detail. They discovered that one chain type, so called linear chains, plays an essential role during a bacterial invasion. Linear ubiquitin chains trigger degradation of bacteria and kick off an inflammatory signaling cascade which results in restricting bacterial proliferation. In addition, the researchers identified the enzyme Otulin as an important regulator capable of limiting this reaction – a very important notion considering the fact that excessive inflammation is one of the major causes of tissue damage following bacterial infection.
Signaling the cells’ need for pathogen defense is just one important role of ubiquitin. The small protein is also involved in development and progression of inflammatory and neurodegenerative diseases as well as of cancer. Until now, however, very little is known about how small errors in the ubiquitin system contribute to these serious human diseases, and how the system can be targeted pharmaceutically.
These new findings pave the way for many follow-up projects which may ultimately lead to novel therapeutic approaches. Very recently, Ivan Dikic obtained one of the prestigious ERC Advanced Grants of 2.5 M € in which he will investigate the role of ubiquitin in modulating the host-pathogen interaction in more detail.
The work of the Frankfurt team is an excellent example for interdisciplinary collaboration and was enabled by funding of several large research networks, e.g. the Cluster of Excellence Macromolecular Complexes, the CRC 1177 on selective autophagy and the LOEWE ubiquitin network. The results are now published in the latest online issue of Nature Microbiology, back-to-back with complementary insights generated by colleagues in Cambridge (UK).
Picture link: www.uni-frankfurt.de/66465906
Captions: 1. Salmonella within a human cell, surrounded by a coat of different ubiquitin chains. Purple represents linear ubiquitin chains, green all ubiquitin chains. Visualized by super-resolution microscopy (dSTORM). Copyright: Mike Heilemann/Ivan Dikic
2. One Salmonella bacterium within a human cell, surrounded by a coat of different ubiquitin chains. Purple represents linear ubiquitin chains, green all ubiquitin chains. Visualized by super-resolution microscopy (dSTORM). Copyright: Mike Heilemann/Ivan Dikic
3. One Salmonella bacterium within a human cell, surrounded by a coat of different ubiquitin chains. The coloured dots represent individual linear ubiquitin chains. Visualized by super-resolution microscopy (3D-dSTORM). Copyright: Mike Heilemann/Ivan Dikic
Publication: van Wijk SJ, Fricke F, Herhaus L, Gupta J, Hötte K, Pampaloni F, Grumati P, Kaulich M, Sou Y, Komatsu M, Greten F, Fulda S, Heilemann M, Dikic I. Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation. Nature Microbiology 2017, doi 10.1038/nmicrobiol.2017.66.
Information: Dr. Kerstin Koch, Institute of Biochemistry II, Faculty 16, University Hospital Frankfurt, Phone +49 (0)69 6301 84250, firstname.lastname@example.org.
Light-inducible microRNA inhibitors used for the first time as local therapeutic agents
FRANKFURT.MicroRNAs are interesting target structures for new therapeutic agents. They can be blocked through synthetic antimiRs. However, to date it was not possible to use these only locally. Researchers at Goethe University Frankfurt have now successfully achieved this in the treatment of impaired wound healing with the help of light-inducible antimiRs.
MicroRNAs are small gene fragments which bond onto target structures in cells and in this way prevent certain proteins from forming. As they play a key role in the occurrence and manifestation of various diseases, researchers have developed what are known as antimiRs, which block microRNA function. The disadvantage of this approach is, however, that the blockade can lead to side effects throughout the entire body since microRNAs can perform different functions in various organs. Researchers at Goethe University Frankfurt have now solved this problem.
The research groups led by Professor Alex Heckel and Professor Stefanie Dimmeler of the Cluster of Excellence Frankfurt Macromolecular Complexes have developed antimiRs that can be activated very effectively over a limited local area by using light of a specific wavelength. To this purpose, the antimiRs were locked in a cage of light-sensitive molecules that disintegrate as soon as they are irradiated with light of a specific wavelength.
As a means of testing the therapeutic effect of these new antimiRs, the researchers chose microRNA-92a as the target structure. This is frequently found in diabetes patients with slow-healing wounds. They injected the antimiRs in the light-sensitive cage into the skin of mice and then released the therapeutic agent into the tissue with the help of light. Together the research groups were able to prove that pinpointed activation of an antimiR against microRNA-92a helps wounds to heal.
“Apart from these findings, which prove for the first time that wound healing can be improved by using antimiRs to block microRNA-92a, our data also confirms that microRNA-92a function is indeed only locally inhibited. Other organs, such as the liver, were not affected”, says Professor Stefanie Dimmeler, underlining the trials’ clinical significance.
The researchers now want to see whether they can also expand the use of light-inducible antimiRs to the treatment of other diseases. In particular they want to examine whether toxic antimiRs can attack tumors locally as well.
Publication: Tina Lucas, Florian Schäfer, Patricia Müller, Sabine A. Eming, Alexander Heckel & Stefanie Dimmeler: “Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice”, in: Nature Communications, publication date, doi: 10.1038/ncomms15162
Further information: Professor Dr. Stefanie Dimmeler, Institute of Cardiovascular Regeneration and Cluster of Excellence Frankfurt Macromolecular Complexes, Excellence Cluster Cardio-Pulmonary System, Faculty of Medicine, Frankfurt University Hospital, Tel.: +49(0)69-798-29475, email@example.com