Experiences with STED microscopy
When Ernst Abbe formulated the theory in 1873 that the maximum resolution of a light microscope was limited to half the wavelength of visible light of about 200 to 350 nanometres, nobody was thinking of observing sturctures of a few nanometres with light microscopes. However, biomedical research scientists are particularly interested in far smaller proteins and intracellular structures. Watching the way proteins and other molecular complexes move, work and interact helps us understand life's processes - how diseases develop and can be treated.
The decisive breakthrough was achieved by the physicist Professor Stefan Hell, now Head of NanoBiophotonics at the Max-Planck Institute in Göttingen, Germany. He invented fluorescence microscopes with which he outwitted Abbe's law. The super high resolution STED microscope is capable of resolving details as small as 20 nanometres. The first STED systems are already in use for researching signal transmisison in nerve cells, for instance. Three scientists who are using STED microscopy for neuroscience research report on their experiences:
Prof. Dr. Stephan Sigrist, Institute of Biology, Freie Universität Berlin and NeuroCluster of Excellence, Charité, Berlin, Germany
Dr. Silvio Rizzoli, European Neuroscience Institute (ENI) and Cluster of Excellence Microscopy at the Nanometer Range at the DFG Research Centre for Molecular Physiology of the Brain (CMPB), Göttingen, Germany
Dr. Gregorz Wilczynski, Laboratory of Molecular and Systemic Neuromorphology, Nencki Institute of Experimental Biology, Warsaw, Poland
What does STED show you that you couldn’t see before?
Sigrist: For the first time, STED brings light into darkness in the field of synaptic proteins. We recognise sub-structures of synapses and are able to localise proteins such as bruchpilot. Bruchpilot plays a key role in synaptic signal transmission in the nerve cells of the Drosophila fly by building up a specific structure there for supporting signal transmission. If the Drosophila fly does not have much bruchpilot, it cannot sustain flight, if it has none at all, it dies. The protein is found in similar form in humans, too, and could be connected with diseases of the nervous system. Studying animals helps to understand the functions of the protein in humans. Understanding biological signal transmission is not only important for science in general. It is probable that synaptic defects trigger a large number of neurodegenerative diseases. In addition, it is almost certain that memory and learning processes are organised at synapses.
Rizzoli: Measuring only 40–50 nm, vesicles, which stock neurotransmitters in the synapsis and release them for signal transmission to the cell membrane, are among the smallest organelles of the nerve cell. Only with the help of STED were we able to localise individual vesicles, which usually occur in groups of 100–300. And contrary to previous assumptions that they hardly moved, we see that they move to and fro all the time, extremely rapidly and seemingly at random. This was inconceivable for us, as signal transmission is a highly complex, controlled process. We managed to record a first live video of these processes. As well as this, our knowledge of the vesicle recycling process at the cell membrane has also been revolutionised by STED. We now know, for example, that after fusing with the cell membrane, vesicle molecules are connected like a drop of oil in water – which facilitates vesicle recycling.
Fig. 3 a–c: The images show various areas in axons of neurons in cell culture. The cells were taken from the hippocampus of a rat (Rattus norvegicus). The synaptic protein Synaptotagmin 1 was labelled with a primary monoclonal mouse antibody and then with a secondary antibody carrying the fluorophore (Atto 647N). In STED mode, the synaptic vesicles can be clearly distinguished as individual points, which is not the case in confocal mode. Courtesy of S. Rizzoli, European Neuroscience Institute (ENI), Göttingen, Germany
Wilczynski: Most synapses are situated on tiny protrusions 200 nm to 2 µm in size called dendritic spines. Their different shapes and sizes are thought to have a crucial influence on signal transmission. Changes of the dendritic spines also play a role in diseases such as epilepsy and the congenital disease Fragile X syndrome. Conflicting evidence has been obtained on dendritic spine form variety so far, and STED enables us to examine it in much more detail than with conventional confocal microscopy and to perform much more analysis than with electron microscopy. With STED we can examine several thousands of dendritic spines in the time it takes us to do 200 to 300 with EM. STED gives our results far higher statistic relevance for a new classification of dendritic spines.
You were one of the first to work with STED. What was it like to see the first images?
Sigrist: Without exaggerating, I can say that I discovered a new world. I immediately realised that STED is a breakthrough for finding answers to our questions and that we had had extremely naïve ideas of what we could see with light microscopy. But, after all, that’s the beauty of science – that new discoveries always raise new questions.
Rizzoli: I well remember the day in 2005 when I took the first photos of vesicles on a STED prototype in Stefan Hell’s laboratory. In those days, it still took five to ten minutes to take a photo – now it only takes 28 milliseconds – and it took us all day to get a good picture. It was fascinating, like opening a new chapter in the book of science. Research scientists must have had similar feelings in the fifties when the first electrophysiological image of a synapsis was produced. Nobody even knew about vesicles then.
Wilczynski: I was naturally delighted with the greater resolution that STED provides. The images are not only a bit sharper, it’s a whole new class of imaging. Actually, I’d expected this new technology to offer higher resolution. But I also found out very quickly that the sample material and the subject of your research play a crucial role for whether you get the most out of the higher resolution.
How do you rate the future significance of STED in life sciences?
Sigrist: Very important indeed, as STED takes us into the realm of protein complexes and therefore gives us a really close up view of life. At present, we are able to resolve structures below the 100 nanometre mark. Professor Hell, who is working on the further development of STED, has already achieved far higher resolutions. If we can use resolutions of a few tens of nanometres, it will be possible to determine with light microscopy whether proteins are close together or further apart. This would constitute a further quantum leap in our understanding of protein functions.
Rizzoli: STED has proved that the former resolution limit can be overcome. Today, everyone who works with conventional high-end resolution wants to work with super high resolution. Meanwhile, many labor-atories are trying to develop new, super high-res techniques. But STED has made the most progress and is the only technology that really works. STED was like a starting pistol for a real technology race. It will be exciting to see what else happens in the next few years.
Wilczynski: I’m quite sure that the significance of super high resolution light microscopy will increase. And I’m also sure that even technologies like STED will continue to improve in terms of resolution. I also see advantages in comparison with EM – morphological examinations of cellular structures that were only possible with complex EM in the past can now be realised much more quickly with STED, as our case shows.