Researchers at the Max Planck Institute for Biophysical Chemistry and the "nanometre-scale microscopy" excellence cluster have successfully used STED microscopy to film the first nanometre-scale video of the inside of a living cell. With up to 28 frames per second and 4 times greater resolution than conventional optical microscopes, they were able to follow the rapid movements of tiny cell building blocks live. This innovation will allow scientists to better understand the processes by which signals are transmitted between nerve cells and to better address other issues in biological and medical research. 

A microscope with particularly sharp focus is required to observe life processes within a cell in detail. Electron microscopes and scanning electron microscopes can provide this but they do not allow scientists to see the inner life of cells. Conventional optical microscopes do not have a high enough resolution. With his Stimulated Emission Depletion (STED) microscope, which was designed as early as 1994 but has only been used since 2000, Stefan Hell, Director of the Max Planck Institute for Biophysical Chemistry in Göttingen was able for the first time to increase the resolution of fluorescence microscopy drastically and hence to lay the foundation for optical microscopy with nanometre-scale resolution.

Hell's innovation was that he was able to overcome the 130-year-old Abbe coefficient in the fluorescence microscope. The novelty of his procedure is that the focus is no longer limited by the wavelength of light. In addition, Hell added a root term to the Abbe formula which now also allows for molecular resolutions. The first commercial STED microscope was launched by Leica in November 2007.

In the past, the Göttingen scientists had already used the STED microscope to see individual protein complexes separately at a distance of 20 to 50 nanometers. These are structures that are 1000 times smaller than a human hair. However, in these snapshots the cells were chemically fixed and hence frozen in their natural living conditions. The long exposure time for an individual image did not yet allow movements to be recorded.

The faster imaging methods that the scientists in Göttingen then developed for STED microscopy now allow them to record movements within a cell directly onto a film. A shorter exposure now enables these movements to be recorded at a resolution of 65 to 70 nanometers.

The object of the researchers' investigations are living nerve cells, or vesicles. These are small sacs containing semiochemicals that are important to the interaction of nerve cells: signals are transmitted between the nerve cells via semiochemicals which are transmitted by the transmitter cells and detected by the recipient cells. Under the microscope, the scientists were able to follow the way in which the fast vesicles move over the entire length of the nerve endings.

For the future, the Göttingen researchers are planning to optimize the STED microscope so that it can deliver even more frames per second, and so that its focus is sharper and it is more sensitive. They also want to use STED microscopy to solve other neurological problems and to obtain a more detailed understanding of physiological processes in cells.

Increase in resolution through STED microscopy using synaptic vesicles. Conventional confocal microscopes are not able to resolve proteins belonging to individual vesicles in the synapse of a nerve cell. STED microscopy, on the other hand, makes these molecules visible - as shown in the protein synaptotagmin in the image on the right. (Photo: S.W. Hell, MPI for Biophysical Chemistry)
(Photo: S.W. Hell, MPI for Biophysical Chemistry)

The focal spot of the isoSTED microscope (bottom left) is almost ball-shaped, unlike in conventional microscopy (top left). A two-color photo (right) allows two mitochondrial proteins, TOM20 (red) and HSP70 (green) to be observed simultaneously in the cell. 
(Picture: Schmidt & Egner /MPI for Biophysical Chemistry)

In the STED microscope (right), vesicles filled with semiochemicals can be observed separately with 3-4 times greater resolutions - unlike in the confocal microscope (left). The arrow shows the vesicle movement within 35 milliseconds
(Photo: S.W. Hell, MPI for Biophysical Chemistry)