A general method for three color STED microscopy with one depletion laser : application to primary neuronal culture

Abstract

Recent advances in the field of optical nanoscopy have equipped molecular neuroscientists with the means to investigate sub-synaptic phenomena with unprecedented spatial resolution. Indeed, early studies employing these methodologies have revealed an extraordinary level of nanoscale spatiotemporal organization of complex and highly dynamic molecular architectures at the pre- and postsynaptic compartments. How exactly these supramolecular complexes are modified through time and experience, though, remains largely unknown. Moreover, the mechanisms by which pre- and postsynaptic protein organization specifies the biochemical and electrophysiological properties of a synapse have yet to be adequately described. The purpose of the current study is thus to develop an inexpensive, accessible platform for super-resolution microscopy (SRM) of synaptic nanostructure.

Though most SRM modalities are too technically and/or financially demanding for widespread use, basic stimulated emission depletion (STED) microscopy systems are now available to researchers at many institutions and require very little training to begin successfully acquiring SR images. One caveat, however, of many such systems is that they may only be capable of SR imaging in two spectral windows due to the inclusion of only one depletion laser in their design. As SR imaging of only two proteins concurrently only promises limited insight into synaptic nanostructure, expansion of these systems' capabilities to localize even one more label would be highly advantageous in these experimental designs and model building. The present study thus develops and optimizes a method to obtain three color SR images on a microscope with a single STED beam.

The unique spectral properties of a long Stokes shift dye, ATTO 490LS, are exploited to add another SR-capable channel without any instrument modifications. This protocol is then applied to image four synaptic proteins simultaneously in primary neuronal culture, with three imaged at STED resolution. More specifically, a tyrosine-phosphorylated subpopulation of the GluA2 glutamate receptor subunit is localized alongside the postsynaptic scaffolding proteins PSD-95 and PSD-93 with the pre-synaptic protein Munc-13 as a confocal landmark. The superior spatial resolutions achieved in this study establish this protocol as an accessible and robust method to introduce an extra SR channel into existing 2-color STED imaging paradigms.