Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

Martin Pauli,Achmed Mrestani,Marzieh Sharifi,Philip Kollmannsberger,Felix Repp,Lydia Kürzinger,Markus Sauer,Manfred Heckmann,Sven Proppert,Mila M Paul,Anna-Leena Sirén

doi:10.1038/s42003-021-01939-z

Abstract

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

Highlights

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity
We focus on large hippocampal mossy fibers boutons (MFBs)[21,22,23] in brain slices (Fig. 1)
We used two-color 2D-direct stochastic optical reconstruction microscopy (dSTORM) with antibodies directed against the presynaptic active zones (AZ) protein Bassoon[31], and the postsynaptic density (PSD) protein Homer 132 (Fig. 2c–e)

Summary

Introduction

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Since synapses differ substantially from one another even in defined structures, e.g., along the ventro-dorsal axis of the hippocampus (a key brain area for learning and memory9) quantitative information with nanometer spatial resolution in large tissue blocks is highly desirable. Self-interference 3D super-resolution microscopy and active point-spread function (PSF) shaping in combination with adaptive optics were introduced to enable 3D localization of emitters in tissue with a thickness of up to 50 μm[15,16]. The latter approach allowed reconstructing super-resolution volumes with an axial depth of several micrometers. Both, photobleaching of fluorophores and inefficient labeling of target proteins due to the restricted penetration of antibodies render quantitative molecular imaging, an intrinsic strength of SMLM14,20, in thick tissue samples complicated

Methods

Results

Conclusion