Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

10.3791/62845 ◽  
2021 ◽  
Author(s):  
Meredith G. Chambers ◽  
Ryan P. McNamara ◽  
Dirk P. Dittmer
Keyword(s):  
Extracellular Vesicles ◽  
Three Dimensions ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

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 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.

scholarly journals Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

2019 ◽  
Vol 116 (37) ◽  
pp. 18423-18428 ◽  
Author(s):  
Huizhong Xu ◽  
Zhisong Tong ◽  
Qing Ye ◽  
Tengqian Sun ◽  
Zhenmin Hong ◽  
...  
Keyword(s):  
Meiotic Chromosome ◽  
Molecular Organization ◽  
Homologous Chromosomes ◽  
Meiotic Chromosomes ◽  
C Terminus ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
20 Nm ◽  
Chromosome Axis

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure’s lateral elements (LEs). While the components of the mammalian chromosome axis/LE—including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2—are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

scholarly journals Mapping Neuronal Connectivity Using Stochastic Optical Reconstruction Microscopy (Storm): The Brainstorm Project

2010 ◽  
Vol 98 (3) ◽  
pp. 214a
Author(s):  
Melike Lakadamyali ◽  
Mark Bates ◽  
Hazen Babcock ◽  
Jeff Lichtman ◽  
Xiaowei Zhuang
Keyword(s):  
Neuronal Connectivity ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

scholarly journals Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ruiheng Li ◽  
Pantelis Georgiades ◽  
Henry Cox ◽  
Sorasak Phanphak ◽  
Ian S. Roberts ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

scholarly journals Endosome–mitochondria interactions are modulated by iron release from transferrin

2016 ◽  
Vol 214 (7) ◽  
pp. 831-845 ◽  
Author(s):  
Anupam Das ◽  
Sagarika Nag ◽  
Anne B. Mason ◽  
Margarida M. Barroso
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Time Lapse ◽  
Iron Release ◽  
Iron Transfer ◽  
Distance Transformation ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Kinetics Of

Transient “kiss and run” interactions between endosomes containing iron-bound transferrin (Tf) and mitochondria have been shown to facilitate direct iron transfer in erythroid cells. In this study, we used superresolution three-dimensional (3D) direct stochastic optical reconstruction microscopy to show that Tf-containing endosomes directly interact with mitochondria in epithelial cells. We used live-cell time-lapse fluorescence microscopy, followed by 3D rendering, object tracking, and a distance transformation algorithm, to track Tf-endosomes and characterize the dynamics of their interactions with mitochondria. Quenching of iron sensor RDA-labeled mitochondria confirmed functional iron transfer by an interacting Tf-endosome. The motility of Tf-endosomes is significantly reduced upon interaction with mitochondria. To further assess the functional role of iron in the ability of Tf-endosomes to interact with mitochondria, we blocked endosomal iron release by using a Tf K206E/K534A mutant. Blocking intraendosomal iron release led to significantly increased motility of Tf-endosomes and increased duration of endosome–mitochondria interactions. Thus, intraendosomal iron regulates the kinetics of the interactions between Tf-containing endosomes and mitochondria in epithelial cells.

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