Source 1primary paper2022Journal of Extracellular Vesicles
Toolkit/Cryo-EM
Cryo-EM
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
These were confirmed by Cryo-EM.
Usefulness & Problems
Why this is useful
Cryo-EM was used to confirm the membrane microdomains detected on extracellular vesicles. In this paper it functions as an orthogonal structural validation method.; orthogonal confirmation of membrane microdomains on extracellular vesicles
Source:
Cryo-EM was used to confirm the membrane microdomains detected on extracellular vesicles. In this paper it functions as an orthogonal structural validation method.
Source:
orthogonal confirmation of membrane microdomains on extracellular vesicles
Problem solved
It provides confirmation for the membrane microdomains inferred from dSTORM imaging.; provides confirmatory evidence for membrane microdomains observed by dSTORM
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It provides confirmation for the membrane microdomains inferred from dSTORM imaging.
Source:
provides confirmatory evidence for membrane microdomains observed by dSTORM
Problem links
provides confirmatory evidence for membrane microdomains observed by dSTORM
LiteratureIt provides confirmation for the membrane microdomains inferred from dSTORM imaging.
Source:
It provides confirmation for the membrane microdomains inferred from dSTORM imaging.
Published Workflows
Objective: To directly visualize individual extracellular vesicles below the diffraction limit in three dimensions and determine whether their surfaces contain molecular microdomains.
Why it works: The workflow combines super-resolution optical imaging to localize nanoscale surface clusters on individual EVs with Cryo-EM as an orthogonal confirmation method for the observed membrane microdomains.
surface clustering of tetraspanins CD81 and CD9 on individual extracellular vesiclesexistence of membrane microdomains on extracellular vesiclesdSTORMCryo-EM
Stages
- 1.3D dSTORM imaging of individual extracellular vesicles(functional_characterization)
This stage addresses the difficulty of directly visualizing sub-diffraction-limit EVs and provides the primary evidence for surface microdomains.
Selection: Direct three-dimensional visualization of individual EVs and localization of surface molecule clusters including CD81 and CD9.
- 2.Cryo-EM confirmation of membrane microdomains(confirmatory_validation)
This stage exists to confirm that the membrane microdomains inferred from dSTORM are supported by an orthogonal method.
Selection: Orthogonal confirmation of membrane microdomains observed in the primary imaging stage.
Steps
- 1.Visualize individual extracellular vesicles in three dimensions by dSTORMprimary imaging assay
To directly visualize individual EVs that are below the diffraction limit of conventional light microscopy.
This is the primary measurement step needed before any structural interpretation or orthogonal confirmation can occur.
- 2.Localize CD81 and CD9 surface clusters on individual extracellular vesiclescluster-localization method
To identify molecule clusters such as CD81 and CD9 on the EV surface and infer membrane microdomains.
Cluster localization follows acquisition of 3D dSTORM data because the microdomain claim depends on analyzing the imaged individual particles.
- 3.Confirm membrane microdomains by Cryo-EMorthogonal validation assay
To confirm the membrane microdomains observed in the dSTORM-based analysis.
Cryo-EM is applied after the dSTORM-based observation because it serves as confirmatory validation of the primary finding.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Target processes
No target processes tagged yet.
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
The abstract supports that Cryo-EM capability is required. It does not provide additional implementation details.; requires Cryo-EM capability
The abstract does not indicate that Cryo-EM was the primary method for localizing CD81 and CD9 clusters in 3D on individual EVs.; the abstract does not describe its resolution, workflow, or comparative throughput in this study
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Individual particle visualization revealed previously underappreciated heterogeneity, structure, and complexity of extracellular vesicles.
Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.
dSTORM was used to visualize individual extracellular vesicles in three dimensions and localize CD81 and CD9 clusters on their surfaces.
Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs.
Cryo-EM confirmed the membrane microdomains observed on extracellular vesicles.
These were confirmed by Cryo-EM.
Approval Evidence
1 source1 linked approval claimfirst-pass slug cryo-em
These were confirmed by Cryo-EM.
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orthogonal validationsupports
Cryo-EM confirmed the membrane microdomains observed on extracellular vesicles.
These were confirmed by Cryo-EM.
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Comparisons
Source-stated alternatives
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
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dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Source-backed strengths
serves as an orthogonal validation method
Source:
serves as an orthogonal validation method
Compared with 3D-dSTORM
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Shared frame: source-stated alternative in extracted literature
Strengths here: serves as an orthogonal validation method.
Relative tradeoffs: the abstract does not describe its resolution, workflow, or comparative throughput in this study.
Source:
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Compared with direct stochastic optical reconstruction microscopy
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Shared frame: source-stated alternative in extracted literature
Strengths here: serves as an orthogonal validation method.
Relative tradeoffs: the abstract does not describe its resolution, workflow, or comparative throughput in this study.
Source:
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Compared with dSTORM
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Shared frame: source-stated alternative in extracted literature
Strengths here: serves as an orthogonal validation method.
Relative tradeoffs: the abstract does not describe its resolution, workflow, or comparative throughput in this study.
Source:
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Compared with imaging
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Shared frame: source-stated alternative in extracted literature
Strengths here: serves as an orthogonal validation method.
Relative tradeoffs: the abstract does not describe its resolution, workflow, or comparative throughput in this study.
Source:
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Compared with imaging surveillance
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Shared frame: source-stated alternative in extracted literature
Strengths here: serves as an orthogonal validation method.
Relative tradeoffs: the abstract does not describe its resolution, workflow, or comparative throughput in this study.
Source:
dSTORM is the primary imaging method contrasted with Cryo-EM as the confirmatory method.
Ranked Citations
- 1.