scholarly journals Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

2018 ◽  
Vol 217 (6) ◽  
pp. 1929-1940 ◽  
Author(s):  
Matthias Spiess ◽  
Pablo Hernandez-Varas ◽  
Anna Oddone ◽  
Helene Olofsson ◽  
Hans Blom ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Focal Adhesions ◽  
Stimulated Emission ◽  
Superresolution Microscopy ◽  
Cell Matrix ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Β1 Integrins ◽  
Activity State ◽  
Optical Reconstruction ◽  
Microscopy Techniques

Integrins are the core constituents of cell–matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation.

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 Patterns of conserved gp120 epitope presentation on attached HIV-1 virions

2017 ◽  
Vol 114 (46) ◽  
pp. E9893-E9902 ◽  
Author(s):  
Meron Mengistu ◽  
Ai-hui Tang ◽  
James S. Foulke ◽  
Thomas A. Blanpied ◽  
Mileidy W. Gonzalez ◽  
...  
Keyword(s):  
Transition State ◽  
Full Range ◽  
Viral Envelope ◽  
Cell Binding ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Viral Particles ◽  
Early Replication ◽  
Optical Reconstruction ◽  
Humoral Responses ◽  
Microscopy Techniques

A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus–cell interactions is now possible via advanced microscopy techniques and reagents. Using such methods, we recently determined that CD4-induced (CD4i) transition state epitopes in the HIV surface antigen, gp120, while not exposed on free particles, rapidly become immunoreactive upon virus–cell binding. Here, we use 3D direct stochastic optical reconstruction microscopy (dSTORM) to show that certain CD4i epitopes specific to transition state structures are exposed across the surface of cell-bound virions, thus explaining their immunoreactivity. Moreover, such structures and their marker epitopes are dispersed to regions of virions distal to CD4 contact. We further show that the appearance and positioning of distal CD4i exposures is partially dependent on Gag maturation and intact matrix–gp41 interactions within the virion. Collectively, these observations provide a unique perspective of HIV during early replication. These features may define unique insights for understanding how humoral responses target virions and for developing related antiviral countermeasures.

scholarly journals Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

2020 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Joshua C. Price ◽  
Raquel Mesquita-Riberio ◽  
Melissa L. Mather ◽  
Izzy Jayasinghe
Keyword(s):  
Single Molecule ◽  
Light Exposure ◽  
Super Resolution ◽  
Observation Time ◽  
Nitrogen Vacancy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nitrogen Vacancy Centers ◽  
Optical Reconstruction ◽  
Living Neurons ◽  
Microscopy Techniques

AbstractStochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the ‘off’-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at ∼ 3-times the speed and ∼ 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

scholarly journals Stochastic optical reconstruction microscopy–based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization

2016 ◽  
Vol 27 (22) ◽  
pp. 3583-3590 ◽  
Author(s):  
Rengasayee Veeraraghavan ◽  
Robert G. Gourdie
Keyword(s):  
Single Molecule ◽  
Quantitative Assessment ◽  
Spatial Organization ◽  
Spatial Association ◽  
Three Dimensions ◽  
Significant Advance ◽  
Relative Localization ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Localization Analysis ◽  
Optical Reconstruction

The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200–300 nm of each other in the xy-plane and within 500–700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy–based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and <50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins.

scholarly journals Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes

2013 ◽  
Vol 24 (13) ◽  
pp. 2112-2123 ◽  
Author(s):  
K. van den Dries ◽  
S. L. Schwartz ◽  
J. Byars ◽  
M.B.M. Meddens ◽  
M. Bolomini-Vittori ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Adaptor Proteins ◽  
Spatial Arrangement ◽  
Superresolution Microscopy ◽  
Ring Model ◽  
Structural Framework ◽  
Dual Color ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Large Clusters

Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell–substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, αMβ2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current “core–ring” model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.

scholarly journals Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM)

2021 ◽  
Author(s):  
Lekha Patel ◽  
David Williamson ◽  
Dylan M Owen ◽  
Edward A K Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Immunological Synapse ◽  
Training Data ◽  
Supplementary Information ◽  
Cellular Structures ◽  
Exact Probability ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Exact Probability Distribution ◽  
Optical Reconstruction

Abstract Motivation Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Results Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Availability and implementation Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary information Supplementary data are available at Bioinformatics online.

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