The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Three-dimensional super-resolution fluorescence microscopy methods such as interferometric photoactivated localization microscopy (iPALM) can localize individual proteins of interest at high resolution (20 nm in XY and 8 nm in Z). These images, however, lack the structural context that could be provided by other imaging methods including electron microscopy. Here, we develop a method that combines iPALM and 3D tomographic transmission electron microscopy (TEM). First, we use iPALM to localize fluorescently-tagged endocytic proteins on the inner plasma membrane of PC12 cells in three dimensions. Next, platinum replicas of these same membranes are imaged with transmission electron microscopy to create tomograms. Finally, both images are combined to create a map of the nanometer scale location of these proteins within their three dimensional cellular context. This technique has the power to build ultra high resolution 3D topographic maps of molecular structures in the context of their native cellular environment.