Synaptic junctions linking individual neurons constitute the fundamental building blocks of our brain. Understanding their inner working is crucial to unravel the mechanisms by which our brain processes information. However, imaging structures at a relevant sub-synaptic level is challenging and has often hampered advances in neuroscience.
Although optical techniques have been key to the development of neuroscience, they suffer from three specific shortcomings: First, conventional optical techniques are limited by diffraction, to resolutions of approximately half the optical wavelength; Second, these optical techniques often come along with toxic effects, induced by prolonged optical illumination (phototoxicity); And third, they do not enable the visualization of cellular nanostructures. To overcome all these limitations, we propose two complementary techniques that rely on protein labeling by fluorescent crystalline nanoparticles (NPs) doped with rare earth ions. The first technique will enable noninvasive optical nanoscale imaging of individual synapses and the second will utilize electron microscopy to visualize the morphology of synaptic structures and the location of individual proteins.
Our preliminary results indicate that single proteins that are labeled with fluorescent NPs can be imaged with nanometer spatial resolution by employing a modified version of stimulated emission depletion (STED). STED enables super-resolution imaging by applying a doughnut shaped laser mode that de-excites all the fluorophores except the ones located at an intensity zero. Even though STED can routinely achieve resolutions of tens of nanometers, it comes at a price of large laser intensities, limiting the range of applications. However, our version of STED will be significantly less phototoxic. After developing our technique, we will utilize it to study the time-resolved distribution of proteins in a forming synapse.
In a parallel experiment, we plan to examine the structural properties of a synapse and the location of different proteins by electron microscopy. We will label individual proteins with our NPs and image them by electron microscopy. Combining the information from cathodoluminescence and electron microscopy will enable the visualization of the exact location of individual proteins labeled with different colors and the morphology of the synapse. As a first application, we will investigate the effect of structural proteins such as neuroligin and neurexin onto the synaptic morphology. The implementation of this project will be done in collaboration with the Südhof’s lab.
These two techniques will help us to shed light on the molecular structure of synapses at an unprecedented level of accuracy and hence will add versatile instruments to the toolbox of neuroscientists.