Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging

Abstract

Imaging neuronal architecture has been a recurrent challenge over the years, and the localization of synaptic proteins is a frequent challenge in neuroscience. To quantitatively detect and analyze the structure of synapses, we recently developed free SODA software to detect the association of pre and postsynaptic proteins. To fully take advantage of spatial distribution analysis in complex cells, such as neurons, we also selected some new dyes for plasma membrane labeling. Using Icy SODA plugin, we could detect and analyze synaptic association in both conventional and single molecule localization microscopy, giving access to a molecular map at the nanoscale level. To replace those molecular distributions within the neuronal three-dimensional (3D) shape, we used MemBright probes and 3D STORM analysis to decipher the entire 3D shape of various dendritic spine types at the single-molecule resolution level. We report here the example of synaptic proteins within neuronal mask, but these tools have a broader spectrum of interest since they can be used whatever the proteins or the cellular type. Altogether with SODA plugin, MemBright probes thus provide the perfect toolkit to decipher a nanometric molecular map of proteins within a 3D cellular context.

Keywords: colocalization; distribution analysis; membrane probe; molecular mapping; super-resolution; synapse.

Fig. 1

Workflow of the Easy SODA protocol available in Icy Software. Colocalization or distant association can be analyzed using the user-friendly Easy SODA protocol that is freely available in Icy Software. This protocol is a graphical programming automatization routine that allows analysis of synaptic proteins’ distribution within neuronal cell shape. Here, neurons are labeled with two proteins (green and cyan channels) that are distributed in clusters. Clusters are segmented using wavelet segmentation through “spot detector” plugin. Cell shape is extracted using a MAP2 stain, and segmentation is done using the “Hierarchical KMeans” plugin. Cluster distribution within the cellular mask is then analyzed through the “SODA” plugin using Ripley’s analysis. Statistical associations are detected if any, and the proportion of associated clusters with their distance is provided with a $p$-value indicating the statistical robustness of the association. If many pictures are analyzed in batch mode, all results can be exported to Excel files. A molecular map is exported for each picture with an association color code. For example, if we take a red-green spot analysis: isolated green spots remain green, green spots associated with red are cyan, isolated red spots remain red, and red spots associated with green are pink. Localization of significant associations is thus visible at a glance over the cell mask (here in deep blue).

Fig. 2

Confocal image (20×) of hippocampal neurons after 2 months in a culture labeled with Cy3.5-MemBright probe reveals dendritic and axonal branch complexity. Scale bar: $100\mu \mathrm{m}$.

Fig. 3

Confocal images (93×) of various cell types labeled with Cy3.5-MemBright probe. (a) 3D rendering of E.coli bacteria incubated overnight with MemBright probes. (b) 3D rendering of HeLa cells incubated 30 min with MemBright. (c) Confocal section of hippocampal neurons incubated several hours with MemBright, revealing intracellular vesicles.

Fig. 4

STORM imaging of hippocampal neurons labeled with Cy3.5-MemBright probe and imaged in conventional widefield microscopy [fire LUT in (a)] and in STORM microscopy (blue spheres in rectangle). (b) Magnification of the plasma membrane 3D STORM image shows the single molecule organization of MemBright all over the plasma membrane. Light blue localizations are closer and deep blue are deeper. (c)–(e) Example of stubby and mushroom dendritic spines in 3D STORM. All the localizations found in (c) (published previously in a different form in Ref. 3) can be used to reconstruct the 3D shape of the spine in a wireframe. This 3D shape can then be used for volumetric estimation or fine measurements of the spine neck.