scholarly journals Label-retention expansion microscopy

2019 ◽  
Author(s):  
Xiaoyu Shi ◽  
Qi Li ◽  
Zhipeng Dai ◽  
Arthur A. Tran ◽  
Siyu Feng ◽  
...  
Keyword(s):  
High Efficiency ◽  
Super Resolution ◽  
Fluorescence Signal ◽  
Signal Loss ◽  
Coated Pits ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Cell Tissue ◽  
Wide Range ◽  
Optical Reconstruction

ABSTRACTExpansion microscopy (ExM) improves the resolution of fluorescence microscopy by physically expanding the sample embedded in a hydrogel1–4. Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples 2–9. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency protein labeling using enzymatic tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved 5 nm resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

scholarly journals Label-retention expansion microscopy

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Xiaoyu Shi ◽  
Qi Li ◽  
Zhipeng Dai ◽  
Arthur A. Tran ◽  
Siyu Feng ◽  
...  
Keyword(s):  
High Efficiency ◽  
Resolving Power ◽  
Fluorescence Signal ◽  
Signal Loss ◽  
Coated Pits ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Cell Tissue ◽  
Wide Range ◽  
Optical Reconstruction

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

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 CTCF-mediated Chromatin Structures Dictate the Spatio-temporal Propagation of Replication Foci

2019 ◽  
Author(s):  
Qian Peter Su ◽  
Ziqing Winston Zhao ◽  
Luming Meng ◽  
Miao Ding ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Super Resolution ◽  
S Phase ◽  
Temporal Organization ◽  
Replication Origins ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Replication Foci ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
Epigenetic Signatures

ABSTRACTMammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically. To understand how replication is regulated at the sub-RD level, we directly visualized the spatio-temporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase using super-resolution stochastic optical reconstruction microscopy (STORM). Importantly, we revealed a hierarchical radial pattern of RFi propagation that reverses its directionality from early to late S-phase, and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a ‘CTCF-organized REplication Propagation’ (CoREP) model. The CoREP model suggests a non-random selection mechanism for replication activation mediated by CTCF at the sub-RD level, as well as the critical involvement of local chromatin environment in regulating replication in space and time.

scholarly journals Mounting Media and Antifade Reagents

2006 ◽  
Vol 14 (1) ◽  
pp. 34-39
Author(s):  
Tony J. Collins
Keyword(s):  
Refractive Index ◽  
Fluorescence Microscopy ◽  
Fluorescence Signal ◽  
Signal Loss ◽  
Biomedical Sciences ◽  
Optical Aberrations ◽  
Wide Range

In the biomedical sciences, samples are mounted in a wide variety of media for examination by microscope. There are a wide variety of mounting media available with a correspondingly wide range of properties. Using the incorrect mounting medium may cause signal loss and optical aberrations; the correct mounting medium avoids such aberrations and preserves fluorescence signal with “anti-fading” properties. This article introduces mounting media for fluorescence microscopy, providing descriptions of their constituents and their properties, as well as accounts of users' experienceMore detailed reviews of antifade reagents have been published by Ono et al. and Longin et al.. Papers describing the effect of refractive index (RI) mismatch have been published by Diaspro et al. and Hell et al..

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

2020 ◽  
Vol 12 (535) ◽  
pp. eaay0071 ◽  
Author(s):  
Zhen Liu ◽  
Quynh P. H. Nguyen ◽  
Qingxu Guan ◽  
Alexandra Albulescu ◽  
Lauren Erdman ◽  
...  
Keyword(s):  
Lung Function ◽  
A Priori ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Defects ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Motile Cilia ◽  
Optical Reconstruction

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

scholarly journals Dissecting the intracellular signalling and fate of a DNA nanosensor by super-resolution and quantitative microscopy

Nanoscale ◽  
2020 ◽  
Vol 12 (28) ◽  
pp. 15402-15413
Author(s):  
Agata Glab ◽  
Alessandro Bertucci ◽  
Fabiana Martino ◽  
Marcin Wojnilowicz ◽  
Alessia Amodio ◽  
...  
Keyword(s):  
Super Resolution ◽  
Intracellular Signalling ◽  
Quantitative Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

The intracellular interactions and fate of a DNA nanosensor were investigated by combining quantitative microscopy and stochastic optical reconstruction microscopy.

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