The Missing Link to the Nanocosm of Life

Superresolution Opens up New Perspectives in Neurobiology

November 02, 2010

Fully understanding the functionality and complexity of the human central nervous system remains one of the major open questions in modern science. In order to address this, understanding the structural architecture of the chemical synapse, a cellular specialization responsible for proper signal transduction and communication between neurons, is of crucial importance. Considering the size of a synapse, e. g., Drosophila NMJ of roughly 500 nm in diameter, it appears logical that, in order to visualize its spatial assembly, the resolution of image acquisition methods needs to be accordingly high. Owing to their diffraction limited resolution, confocal and widefield fluorescence microscopes cannot properly display subsynaptic organization. Stimulated emission depletion microscopy (STED) can be the method to overcome this barrier.

Revealing biological nanostructures

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

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

By dissecting the molecular composition of the synapse and its architecture, one can gather valuable information concerning the machinery of signal transduction and modulation. It is not surprising that the more that is known about the synapse, the higher the demand for visualizing small nuances that  impact the structure's functionality. Most synapses, though, are tiny cellular specializations. In order to explore the synaptic architecture using simple light microscopy, an image resolution that displays structures down to the molecular level cannot be achieved.

One widely used method for reaching higher resolution is to use an electron microscope (EM), which achieves higher resolution by irradiating the probes with considerably smaller wavelengths than used in light microscopy. Through EM and in combination with tomographic image processing, synaptic structures can be visualized with a resolution of only a few nanometers, which is many times higher than any light microscopy technique. A drawback is that EM often involves quite elaborate dehydration and contrasting procedures. Even though very small structures are nicely displayed, attributing the visualized structures to the localization of one or more proteins via immuno-labeling remains tricky. Furthermore, time-consuming difficulties arise when high resolution images are needed from bigger or thicker samples, and several EM slices need to be merged or reconstructed. 

Recent advances in light microscopy, such as the development of STED microscopy [1], greatly contribute to this issue by offering a revolutionary simple method of fluorescence visualization with image resolution ranging down to 30 nm, and creating the fully new concept of nanobiophotonics.

Fig. 1: Presynaptic T-bar structure depicted by different imaging techniques. (A) NMJ labeled with two antibodies for BRP, either for the C-term (Nc82) or N-term. Because of its spherical structure of single subdivisions of the Drosophila NMJ (boutons), single synapses (see inset) show a segregation of the BRPN-term label toward the outside of the bouton. As in these regions the synaptic membrane lies perpendicular to the focal plane, a polarized orientation of BRP from the membrane to cytoplasm may be implicated. (B) Same structures now imaged with STED (green) and a confocal reference (red). In these images an architectural arrangement was described for BRP that depicts striking similarities to the T-bar observed in electron micrographs (C).
Three different imaging methods showing different degrees of information. Sample: Presynaptic T-bar structure, NMJ labeled with two antibodies for BRP, either for the C-term (Nc82) or N-term. Because of its spherical structure of single subdivisions of th

Contributions of STED to neurobiology

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 [2]. 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 [7].

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.

Fig. 2: Single synapses of different sizes show considerable structural rearrangement. STED images of GFP show DLiprin-alphaGFP as discrete dots arranged around the synapse core labeled by BRPNc82 (magenta), ranging from one to two dots at small, freshly assembled synapses and from four to five dots at mature ones. These architectural features weren‘t stipulated in EM pictures nor in confocal images, demonstrating the importance of STED analysis for structural characterization at the Drosophila neuromuscular junction. Scale bar: 250 nm.
Six examples of synapses that show considerable structural rearrangement. Synapse core labeled by BRPNc82 and imaged confocal superimposed with Liprin-GFP superresolution images recorded with Leica TCS STED.

Superresolution in live cell imaging

Live cell imaging, though, is where STED microscopy shows its most considerable strengths. Understanding small structural changes, protein localization, turn-over rate or redistribution in live cells, especially during neuronal activity, is crucial for the characterization of synapses. This not only holds true for neurobiological research, but concerns many fields in biological and medical sciences. Developments in STED technology, which were first limited to‚ above-average bright and stable fluorescent dyes, such as Atto®594 and Atto®647N, more recently allowed  the visualization of fluorescent proteins in live specimens with both recently developed far-red fluorescent proteins 8 and commonly used markers such as EGFP and EYFP [2, 9, 10, 11].

With these improvements, the gain of resolution previously limited to fixed tissue, can now be achieved in live specimens. Also, STED is the most straightforward technique to visualize dynamic protein reorgan-ization, since it can penetrate tissue considerably (15–20 µm is typical), allows fast image acquisition, and doesn‘t depend on stochastic post-processing and reconstruction. STED, therefore, opens new possibilities of data acquisition including time-lapse and FRAP experiments. With the application of this method a whole new range of questions regarding dynamic aspects may be addressed, enabling superresolution for live cell imaging.

Fig. 3: Live cell imaging with STED CW. Consecutive STED images (single confocal slices) of CitrineDLiprin-alpha in intact live Drosophila larvae demonstrate, in principle, the possibilities for time lapse and FRAP experiments.
Superresolution Time Lapse Sequence with Leica TCS SP5 STED microscope system. Citrine-DLiprin in intact live Drosophila larvae demonstrate the proof of principle for time lapse and FRAP experiments with Superresolution STED in living samples.

References

  1. Hell SW, Dyba M and Jakobs S: Concepts for Nanoscale Resolution in Fluorescence Microscopy. Current Opinions in Neurobiology 14 (2004) 599–609.
  2. Westphal V et al.: Video-rate Far-field Optical Nanoscopy Dissects Synaptic Vesicle Movement. Science 320 (2008) 246–9.
  3. Willig KI et al.: STED Microscopy Reveals that Synaptotagmin Remains Clustered after Synaptic Vesicle Exocytosis. Nature 440 (2006) 935–939.
  4. Kittel RJ. et al.: Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release. Science 312 (2006) 1051–1054.
  5. Fouquet W et al.: Maturation of Active Zone Assembly by Drosophila Bruchpilot. The Journal of Cell Biology 186 (2009) 129–145.
  6. Owald D. et al.: A Syd-1 Homologue Regulates Pre- and Postsynaptic Maturation in Drosophila. The Journal of Cell Biology 188 (2010) 565–79.
  7. Regus-Leidig H et al.: Early Steps in the Assembly of Photoreceptor Ribbon Synapses in the Mouse Retina: the Involvement of Precursor Spheres. The Journal of Comparative Neurology 512 (2009) 814–24.
  8. Morozova KS. et al.: Far-red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy. Biophysical Journal 99 (2010) L13–5.
  9. Hein B, Willig KI and Hell SW: Stimulated Emission Depletion (STED) Nanoscopy of a Fluorescent Protein-labeled Organelle Inside a Living Cell. Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 14271–6.
  10. Nagerl UV et al.: Live-cell Imaging of Dendritic Spines by STED Microscopy. Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 18982–7.
  11. Nägerl UV and Bonhoeffer T: Imaging Living Synapses at the Nanoscale by STED Microscopy. The Journal of Neuroscience – the official journal of the Society for Neuroscience 30 (2010) 9341–6.

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