Without oxygen, cells cannot survive. If the oxygen supply drops, for example due to a heart attack, long-term damage may result. However, just how serious such damage really is can only be assessed hours or even days later. For the first time now, and using light microscopy, scientists in Münster have observed reduced oxygen supply directly in individual cells. This was technically not feasible before, because there was no possibility to indicate an acute lack of oxygen using so-called reporters. Such a reporter was now developed by a research team led by Prof. Friedemann Kiefer and Prof. Michael Schäfers as part of the Cells-in-Motion Cluster of Excellence (CiM) and Collaborative Research Centre 656, “Molecular Cardiovascular Imaging” at Münster University.
The reporter, consisting of protein, encoded by a special DNA molecule, a so-called DNA construct, works in such a way that fluorescent molecules are produced in cell with insufficient oxygen. This makes them “light up” and enables scientists to see in the microscope what is happening where. The Münster scientists used a reporter protein from an eel to indicate hypoxia – the first time this has been done. The trick here is that, unlike other fluorescent proteins often used, this special protein does not need oxygen in order to be able to fluoresce. This is crucial if scientists want to visualize an acute lack of oxygen under the microscope, because it enables them to still see fluorescence in the case of acute hypoxia. “So this means that if a cell asphyxiates, a green fluorescent light goes on and we can see how dramatic the hypoxia is,” explains Friedemann Kiefer, a biochemist engaged in research at the Max Planck Institute for Molecular Biomedicine in Münster.
Another novelty is that this reporter is particularly sensitive for the oxygen supply of a cell. If a cell recovers because it is again receiving sufficient oxygen, the fluorescence decreases. As the researchers use a variety of reporters for their experiments, other reporters light up in this state of recovery. “With the methods used up to now, all we could do was verify that there had actually been a reduction in the oxygen supply,” says Kiefer. “Using the various reporters, we can now observe a dynamic process and can carry out very accurate research into the long-term consequences of hypoxia.” This means that scientists can not only assess whether a cell was hypoxic previously, but also whether the condition will continue.
As the new reporter indicates a lack of oxygen with extreme sensitivity, there are now entirely new possibilities for its use in science. The combination of the eel protein and light microscopy enables pathologies and their development to be visualized. This has already been possible for some while in a larger format. Using so-called positron emission tomography (PET), doctors and scientists can draw conclusions about illnesses or see, for example, where an infection is developing in the body. Similar images are now also possible using optical microscopy with the new reporter. “This means closing a gap between molecular imaging and microscopy,” says Michael Schäfers, a physician who is also Co-Coordinator of the CiM Excellence Cluster. “This is a great success for imaging processes in research, as well as for use in hospitals.”
Erapaneedi R, Belousov VV, Schäfers M, Kiefer F. (2016): A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level. Embo J;35: 102-113. DOI 10.15252/embj.201592775. Abstract