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) 
