Decifering organelles and synaptic molecular assemblies using quantitative super-resolution 3D microscopy
Elucidating molecular organization and synapse functions requires localizing precisely single or aggregated molecules and analyzing their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve standard co-localization techniques (Lagache et al, Nature Comm 2018). Our method considers neuronal geometry to provide statistical maps of isolated and associated (coupled) molecules. SODA can be used with three-color structured-illumination microscopy (SIM) images to statistically characterize spatial organization of thousands of protein clusters or vesicles. As this methods do not rely on physical overlaps, SODA can be used to detect indirect molecular association in conventional microscopy or even in super resolution microscopy where higher resolution prevent the use of conventional overlay methods.
We used SODA with three-color SIM images of hippocampal neurons, and statistically characterized spatial organization of thousands of synapses. We show that presynaptic synapsin is arranged in asymmetric triangle with the 2 post-synaptic markers homer and PSD95 indicating a deeper localization of homer. As a proof of concept, we then imaged presynaptic glutamatergic terminals with 3D-STORM microscopy and analyzed the coupling between more than 180,000 localizations of vesicular Glutamate Transporter (VGLUT) and Synapsin molecules inside synaptic boutons. We evaluated with SODA that each Synapsin or VGLUT localization is at a mean distance of 52 ± 0,04 nm. We will show how our plugin SODA can also be useful to decipher molecular distance between protein within specific organelles like ER using 3D STED (Gallo et al, JCS 2020) or golgi apparatus in STORM (Sikora et al. Biology Open 2021).
To reconstitute molecular maps within cellular shape, we recently developed new membrane probes call the MemBright family (Collot 2019, on the cover of Cell Chemical Biololgy – April). MemBright is a family of six cyanine based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM. These probes are compatible with long-term live-cell imaging and immunostaining both in culture and tissue without any use of transfection or transgenic animals. We will show how MemBright probes can be used in 3D multicolor dSTORM imaging to unravel the plasma membrane structures of synaptic terminals or tiny dendritic spines protrusions in combination with immunostaining of synaptic proteins.