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.

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Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.super-resolution imaging | synaptonemal complex | dSTORM | meiosis | average position determination T he synaptonemal complex (SC) is a well-preserved meiosisspecific protein complex among different species (1, 2). As revealed by transmission electron microscopy (TEM), when fully assembled, SCs are 200-nm-wide, ribbon-like structures that extend all along a chromosome bivalent (3, 4). SCs have a characteristic ladder-like organization that is highly conserved through evolution and consists of two lateral elements (LEs), at which chromatin of homologous chromosomes is attached, and a central region (CR). The CR holds the homologous chromosomes together and is made up of numerous transversal filaments (TFs) and the central element (CE).At present, seven protein components of the synaptonemal complex have been identified in mammals, namely, the LE proteins SYCP2 and SYCP3 (5, 6); the TF protein SYCP1 (7); and the CE-specific proteins SYCE1, SYCE2, SYCE3, and TEX12 (8-10) (Figs. S1 and S2). However, in addition to the identification of SC protein components and the investigation of interaction partners, the establishment of a model of the molecular architecture remains indispensable for the understanding of its function and assembly process. Due to the resolution limit of conventional fluorescence microscopy, information about the molecular organization of SCs has been mainly obtained by immunogold EM (11). In standard immunogold EM preparations, gold particles are localized with nanometer resolution and the localization precision is mainly limited by the size of the primary and secondary IgG antibodies and the gold signal density (12-14). However, sample preparation is time-consuming and quantitative analysis of the signal is tedious because of the low binding efficiency of goldlabeled antibodies. Because the structural resolution is also determined by the labeling density (15), immunogold EM cannot visualize the different SC proteins as continuous structures. Therefore, the construction of ...

Crystal clear: The authors introduce a miniaturized localization microscopy setup based on cost-effective components. They demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.