TY  - JOUR
A1  - Markert, Sebastian Matthias
A1  - Britz, Sebastian
A1  - Proppert, Sven
A1  - Lang, Marietta
A1  - Witvliet, Daniel
A1  - Mulcahy, Ben
A1  - Sauer, Markus
A1  - Zhen, Mei
A1  - Bessereau, Jean-Louis
A1  - Stigloher, Christian
T1  - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome
JF  - Neurophotonics
N2  - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
KW  - caenorhabditis elegans
KW  - localization micoscopy
KW  - fluorescent-probes
KW  - junction proteins
KW  - resolution limit
KW  - direct stochasticoptical reconstruction microscopy
KW  - structured illumination microscopy
KW  - correlative light and electron microscopy
KW  - gap junction
KW  - neural circuits
KW  - nervous-system
KW  - image data
KW  - reconstruction
KW  - innexins
KW  - super-resolution microscopy
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292
VL  - 3
IS  - 4
ER  - 
TY  - JOUR
A1  - Proppert, Sven
A1  - Wolter, Steve
A1  - Holm, Thorge
A1  - Klein, Theresa
A1  - van de Linde, Sebastian
A1  - Sauer, Markus
T1  - Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging
JF  - Optics Express
N2  - In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
KW  - three-dimensional microscopy
KW  - fluorescence microscopy
KW  - medical and biological imaging
KW  - superresolution
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119730
SN  - 1094-4087
VL  - 22
IS  - 9
ER  - 
TY  - JOUR
A1  - Pauli, Martin
A1  - Paul, Mila M.
A1  - Proppert, Sven
A1  - Mrestani, Achmed
A1  - Sharifi, Marzieh
A1  - Repp, Felix
A1  - Kürzinger, Lydia
A1  - Kollmannsberger, Philip
A1  - Sauer, Markus
A1  - Heckmann, Manfred
A1  - Sirén, Anna-Leena
T1  - Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections
JF  - Communications Biology
N2  - 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 mu m, 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 mu m 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. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.
KW  - mossy fiber synapses
KW  - CA3 pyrimidal cells
KW  - CA2+ channels
KW  - active zone
KW  - hippocampal
KW  - release
KW  - plasticity
KW  - proteins
KW  - platform
KW  - reveals
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259830
VL  - 4
ER  -