Fully understanding the functionality and the complexity of the human central nervous system remains as one of the major open questions in modern science. The Drosophila neuromuscular junction (NMJ) is a widely used and acknowledged model for the analysis of synapse structure and assembly. When considering the size of a Drosophila NMJ synapse of about 500 nm in diameter it appears logical that in order to visualize its spatial architecture the resolution of image acquisition methods needs to be accordingly high. Owing to their diffraction limited resolution, confocal and wide field microscopes cannot properly display subsynaptic organization in a satisfying manner. This problem may be solved with STED microscopy.

In the past decades, many significant leaps have been made to reveal the secrets of the human central nervous system. The most prominent of these findings involved the characterization of the chemical synapse, which describes a highly specialized region in neurons. Here electrical impulses are translated into a chemical signal in order to transmit the information within a neuronal circuit or to a specific target cell. Through this signal translation, information can rapidly be modulated and processed by either strengthening of weakening the transmission efficiency.

Therefore, synapses and their corresponding neuronal networks are thought to coordinate adequate responses to the environmental stimulus, learn and store information from past experiences and finally form the basis for processes like cognition. The answer of how this is possible lies deeply buried beneath the complexity of the neuronal wiring and the multitude of synaptic proteins with their numerous functions.

Revealing Biological Nanostructures

By unraveling the molecular composition of the synapse and its architecture at a given sites, one can gather valuable information concerning the machinery of signal transduction and modulation. Therefore it is not surprising that the demand in the scientific community for visualizing such structures is constantly growing. Most synapses, though, are very small structures and in many cases in order to explore the synaptic architecture, one requires an image resolution capable of displaying structures down to the molecular level.

One common method for reaching this resolution is to use electron microscopes, which achieve higher resolution by irradiating the probes with considerably smaller wavelengths than normally used in light microscopy.

One disadvantage of this procedure is the fact that it often involves quite elaborate dehydration and contrasting procedures, and even though very small structure are nicely displayed, attributing the protein localization to visualized structures in electron micrographs via immuno-labellings remains tricky.

Recent advances in light microscopy research, especially the invention of the STED technology [1] and the development of commercial STED systems, however, greatly contributed to this issue by developing a revolutionary simple method of fluorescence visualization with the image resolution ranging down to 30nm, thereby creating the fully new concept of nanobiophotonics.

New Insights in Presynaptic Structures

In the lab of Prof. Dr. Stephan Sigrist [2,3] studies were performed on Drosophila NMJ to analyze the synapse structure and assembly. Presynaptic electron dense structures named "T-bars" (owing to their characteristic shape in electron micrographs) were shown to be composed of Bruchpilot (BRP) a member of the conserved CAST/ERC protein family. BRP is thought to play a role as a presynaptic scaffolding protein implicated in proper signal transduction.

Through the application of the STED technology, in a synergic combination with already established imaging techniques, valuable information concerning the architecture of the roughly 250 nm size T-bar and adjacent structures could be obtained. STED microscopy thereby revealed a more precise localization of synaptic proteins, which until then were unrecognizable by conventional confocal imaging (Fig. 1), especially when combined with structural information obtained in electron micrographs.

Thus, STED could be described as a "missing link" between confocal and electron microscopy. In addition, the possibility of generating images in large scale, due to its simple methodology, helped to certify certain assumptions during data acquisition (as for the polarized, elongated orientation of BRP, see Fig. 1), turning them to solid statements. Eventually, the characterization of the synaptic architecture now opens new possibilities by applying the powerful genetic tools of Drosophila in order to disrupt these structures through the generation of mutant animals. By analyzing these mutants, important information concerning the neuronal processing will be available, which in future may contribute for a general understanding of the central nervous system functions and how complex processes as learning and memory are accomplished.