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

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Martin Pauli ◽  
Mila M. Paul ◽  
Sven Proppert ◽  
Achmed Mrestani ◽  
Marzieh Sharifi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mossy Fiber ◽  
Three Dimensional ◽  
Brain Regions ◽  
Molecular Organization ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Axial Scanning ◽  
Single Molecule Localization Microscopy

AbstractRevealing 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.

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

2019 ◽  
Author(s):  
Martin Pauli ◽  
Mila M. Paul ◽  
Sven Proppert ◽  
Marzieh Sharifi ◽  
Felix Repp ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mossy Fiber ◽  
Three Dimensional ◽  
Brain Regions ◽  
Molecular Organization ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Axial Scanning ◽  
Single Molecule Localization Microscopy

ABSTRACTRevealing the molecular organization of anatomically precisely defined brain regions is necessary for the refined understanding of synaptic plasticity. Although, three-dimensional (3D) single-molecule localization microscopy can provide the required molecular resolution, single-molecule 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 aberration-free volumetricdirectstochastic 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 enables 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.

scholarly journals About samples, giving examples: Optimized Single Molecule Localization Microscopy

2019 ◽  
Author(s):  
Angélique Jimenez ◽  
Karoline Friedl ◽  
Christophe Leterrier
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Full Potential ◽  
Coated Pits ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nanoscale Topography ◽  
Biological Discovery ◽  
Optical Reconstruction ◽  
Single Molecule Localization Microscopy

AbstractSuper-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

scholarly journals A cross–nearest neighbor/Monte Carlo algorithm for single molecule localization microscopy defines interactions between p53, Mdm2, and MEG3

2019 ◽  
Author(s):  
Nicholas C. Bauer ◽  
Anli Yang ◽  
Xin Wang ◽  
Yunli Zhou ◽  
Anne Klibanski ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Single Molecule ◽  
Nearest Neighbor ◽  
Monte Carlo Algorithm ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Cellular Context ◽  
Optical Reconstruction ◽  
Suppress Cell Proliferation ◽  
Single Molecule Localization Microscopy

AbstractThe functions of long noncoding (lnc)RNAs such as MEG3 are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with Mdm2. To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule localization microscopy (SMLM) technique to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross–nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the binding between MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.

scholarly journals Spatial filter and its application in three-dimensional single molecule localization microscopy

2020 ◽  
Vol 8 (2) ◽  
pp. 025008
Author(s):  
Xiaoming Fan ◽  
Johnny Hendriks ◽  
Maddalena Comini ◽  
Alexandros Katranidis ◽  
Georg Büldt ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Spatial Filter ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Three-dimensional biplane spectroscopic single-molecule localization microscopy: erratum

Optica ◽  
2019 ◽  
Vol 6 (10) ◽  
pp. 1374
Author(s):  
Ki-Hee Song ◽  
Yang Zhang ◽  
Gaoxiang Wang ◽  
Cheng Sun ◽  
Hao F. Zhang
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

2020 ◽  
Vol 6 (16) ◽  
pp. eaay8271 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Microscopy ◽  
Direct Distance ◽  
Time Drift ◽  
Localization Precision ◽  
20 Nm ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

scholarly journals Three dimensional drift control at nano-scale in single molecule localization microscopy

2020 ◽  
Vol 28 (22) ◽  
pp. 32750
Author(s):  
Xiaoming Fan ◽  
Thomas Gensch ◽  
Georg Büldt ◽  
Yuanheng Zhang ◽  
Zulipali Musha ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Localization Microscopy ◽  
Nano Scale ◽  
Single Molecule Localization Microscopy ◽  
Drift Control

scholarly journals Analyzing engineered point spread functions using phasor-based single-molecule localization microscopy

2020 ◽  
Author(s):  
Koen J.A. Martens ◽  
Abbas Jabermoradi ◽  
Suyeon Yang ◽  
Johannes Hohlbein
Keyword(s):  
Saddle Point ◽  
Single Molecule ◽  
Software Package ◽  
Double Helix ◽  
Three Dimensional ◽  
Fourier Plane ◽  
Point Spread Functions ◽  
Localization Microscopy ◽  
Point Spread ◽  
Single Molecule Localization Microscopy

The point spread function (PSF) of single molecule emitters can be engineered in the Fourier plane to encode three-dimensional localization information, creating double-helix, saddle-point or tetra-pod PSFs. Here, we describe and assess adaptations of the phasor-based single-molecule localization microscopy (pSMLM) algorithm to localize single molecules using these PSFs with sub-pixel accuracy. For double-helix, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations, while for saddle-point or tetra-pod, a novel phasor-based deconvolution approach is used. The pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores. pSMLM substantially improves the localization rate by a factor of 2 - 4x on a standard CPU, with 1-1.5·104 (double-helix) or 2.5·105 (saddle-point/tetra-pod) localizations/second.

scholarly journals Symmetrically-dispersed spectroscopic single-molecule localization microscopy

2019 ◽  
Author(s):  
K. Song ◽  
Y. Zhang ◽  
B. Brenner ◽  
C. Sun ◽  
H. F. Zhang
Keyword(s):  
Spectral Analysis ◽  
Single Molecule ◽  
Particle Tracking ◽  
Three Dimensional ◽  
Spatial Localization ◽  
Single Particle Tracking ◽  
Localization Microscopy ◽  
First Time ◽  
Spectral Channels ◽  
Single Molecule Localization Microscopy

AbstractSpectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.

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