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 Toward Sub-Diffraction Imaging of Single-DNA Molecule Sensors Based on Stochastic Switching Localization Microscopy

Sensors ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 6667
Author(s):  
Seungah Lee ◽  
Indra Batjikh ◽  
Seong Ho Kang
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Single Molecule Level ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Wide Range ◽  
Nanoscale Topography ◽  
Diffraction Imaging ◽  
Optical Reconstruction

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

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 DNA-Based Super-Resolution Microscopy: DNA-PAINT

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 621 ◽  
Author(s):  
Daniel Nieves ◽  
Katharina Gaus ◽  
Matthew Baker
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Cutting Edge ◽  
Cellular Proteins ◽  
Localization Microscopy ◽  
Nanoscale Topography ◽  
History Of ◽  
Super Resolution Microscopy ◽  
Fluorescent Ligand ◽  
Single Molecule Localization Microscopy

Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.

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.

Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images

2018 ◽  
Vol 87 (1) ◽  
pp. 965-989 ◽  
Author(s):  
David Baddeley ◽  
Joerg Bewersdorf
Keyword(s):  
Imaging Modality ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Biological Insight ◽  
Biological Discovery ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy ◽  
Fluorescent Molecules

Super-resolution optical imaging based on the switching and localization of individual fluorescent molecules [photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc.] has evolved remarkably over the last decade. Originally driven by pushing technological limits, it has become a tool of biological discovery. The initial demand for impressive pictures showing well-studied biological structures has been replaced by a need for quantitative, reliable data providing dependable evidence for specific unresolved biological hypotheses. In this review, we highlight applications that showcase this development, identify the features that led to their success, and discuss remaining challenges and difficulties. In this context, we consider the complex topic of defining resolution for this imaging modality and address some of the more common analytical methods used with this data.

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 Single-molecule Photoswitching and Localization

2011 ◽  
Vol 64 (5) ◽  
pp. 503 ◽  
Author(s):  
Sebastian van de Linde ◽  
Steve Wolter ◽  
Markus Sauer
Keyword(s):  
Single Molecule ◽  
Optical Resolution ◽  
Super Resolution ◽  
Image Plane ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
20 Nm

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

scholarly journals Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

2019 ◽  
Vol 16 (5) ◽  
pp. 387-395 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Fight Club ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

scholarly journals Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

2018 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Super Resolution ◽  
Large Set ◽  
Holistic View ◽  
Localization Microscopy ◽  
Fight Club ◽  
Community Effort ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

ABSTRACTWith the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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