Conventional fluorescent microscopy is perfectly suited for analysis of a biological specimen, since the localization of fluorescent dyes is easily assessed in both fixed and living specimens. STED goes one step further by enabling the detailed discrimination of even smaller cellular organelles and sub-compartments. In neurobiology many considerable achievements have been made, as described in a few examples below:
Synaptic vesicles (50–80 nm) are transport units used by the cell to harvest neurotransmitters, which on demand, are fused with the presynaptic membrane and release their content into the synaptic cleft. Understanding the process of how such vesicles are formed, transported, and docked to the proper release site and how the endo/exocytosis vesicle recycling works is hugely important to the scientific community. Recently, video rate STED imaging of live specimens was used to describe vesicle mobility along axons . With the help of STED, the transport of vesicles was described more precisely, detecting even small changes in speed and direction otherwise unrecognizable in conventional image acquisition. In other experiments3 the localization of a synaptic vesicle’s associated protein (Synaptotagmin) was characterized upon vesicle fusion. Their findings contributed to an overall understanding of how vesicle-specific proteins may be retrieved from the plasma membrane during endocytosis.
Temporal aspects of how single components of the synapse are incorporated into the protein matrix throughout synapse maturation, e.g. via synaptic precursor vesicles, are not yet fully understood. Studies on the Drosophila NMJ were performed to analyze the synapse structure and assembly [4, 5, 6]. Presynaptic electron dense structures named “T-bars” (owing to their characteristic shape in electron micrographs) were shown to comprise Bruchpilot (BRP). BRP is thought to play a role in signal transduction by acting as a presynaptic scaffolding protein. Through the application of STED technology, in a synergistic combination with established imaging techniques, valuable information concerning the architecture of the roughly 250 nm size T-bar and adjacent structures was obtained (Figure 1). Similar studies as in Drosophila were performed on murine retina cells, where the composition of presynaptic proteins associated to precursor vesicles, which are thought to promote synaptogenesis, was described .
In the examples above, STED microscopy revealed a very precise distribution of fluorescently-tagged synaptic proteins, which until then were unrecognizable via conventional confocal imaging. When compared to data from electron micrographs, a whole new set of information was retrieved. But unlike EM, STED, due to its simple methodology, allowed image acquisition not only on an uncomplicated and quick fashion, but also in a larger scale, thereby assisting in a more extensive understanding of the synaptic structure and its impact on the signal transduction (Figure 2). Thus, STED can be generally described as a “missing link” between confocal and electron microscopy.
The STED findings concerning the characterization of the synaptic architecture broaden our understanding of the synapse function, which contributes to the general picture of how the central nervous system works and how complex processes such as learning and memory are accomplished.