Source 1review2023Nature Methods
Toolkit/cryo-electron tomography
cryo-electron tomography
Also known as: cryo-ET
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).
Usefulness & Problems
Why this is useful
Cryo-EM/ET is used here to characterize the immature HTLV-1 CA lattice in virus particles. The abstract specifically attributes detection of lattice curvature and CA-to-membrane distance heterogeneity to cryo-ET.; characterizing immature virus particle capsid lattice morphology; detecting lattice heterogeneity including curvature and membrane spacing variation; Cryo-ET is used to study structures within the near-native cellular interior and surroundings. The abstract contrasts it with isolated-molecule cryo-EM by emphasizing cellular-context structural information.; in situ structural studies of the near-native cellular interior; obtaining structural information in the cellular environment
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Cryo-EM/ET is used here to characterize the immature HTLV-1 CA lattice in virus particles. The abstract specifically attributes detection of lattice curvature and CA-to-membrane distance heterogeneity to cryo-ET.
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characterizing immature virus particle capsid lattice morphology
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detecting lattice heterogeneity including curvature and membrane spacing variation
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Cryo-ET is used to study structures within the near-native cellular interior and surroundings. The abstract contrasts it with isolated-molecule cryo-EM by emphasizing cellular-context structural information.
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in situ structural studies of the near-native cellular interior
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obtaining structural information in the cellular environment
Problem solved
It enables direct structural observation of immature particle morphology and lattice organization that are not captured by lower-resolution descriptions.; provides structural characterization of immature HTLV-1 capsid lattice organization; It helps recover structural information in the cellular environment where macromolecules perform native functions. This addresses interactions that may be lost during isolation.; addresses loss of cellular-context structural information that can occur during isolation of macromolecules
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It enables direct structural observation of immature particle morphology and lattice organization that are not captured by lower-resolution descriptions.
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provides structural characterization of immature HTLV-1 capsid lattice organization
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It helps recover structural information in the cellular environment where macromolecules perform native functions. This addresses interactions that may be lost during isolation.
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addresses loss of cellular-context structural information that can occur during isolation of macromolecules
Problem links
addresses loss of cellular-context structural information that can occur during isolation of macromolecules
LiteratureIt helps recover structural information in the cellular environment where macromolecules perform native functions. This addresses interactions that may be lost during isolation.
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It helps recover structural information in the cellular environment where macromolecules perform native functions. This addresses interactions that may be lost during isolation.
provides structural characterization of immature HTLV-1 capsid lattice organization
LiteratureIt enables direct structural observation of immature particle morphology and lattice organization that are not captured by lower-resolution descriptions.
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It enables direct structural observation of immature particle morphology and lattice organization that are not captured by lower-resolution descriptions.
Published Workflows
Objective: Characterize the structure and morphology of the HTLV-1 immature capsid lattice and assess whether IP6 is required for immature particle assembly and lattice formation.
Why it works: The workflow combines higher-resolution SPA reconstruction to identify lattice interfaces with cryo-ET analysis to capture particle-level heterogeneity that may not be represented by a single reconstruction.
trimeric NTD inter-hexamer interfacedimeric CTD inter-hexamer interfaceIP6 dispensability for immature assembly and lattice formationcryo-electron microscopy and tomographysingle particle analysis
Stages
- 1.High-resolution lattice reconstruction(functional_characterization)
This stage provides the high-resolution reconstruction needed to infer the specific NTD and CTD inter-hexamer interfaces that stabilize the lattice.
Selection: Resolve the immature CA lattice structure at high resolution to identify stabilizing interfaces.
- 2.Tomographic heterogeneity analysis(secondary_characterization)
This stage captures heterogeneity in immature particle morphology that complements the higher-resolution reconstruction.
Selection: Analyze immature particles for morphological heterogeneity beyond the single reconstructed lattice model.
Steps
- 1.Resolve immature CA lattice by SPAassay method and structural subject
Obtain a high-resolution reconstruction of the immature HTLV-1 CA lattice.
High-resolution reconstruction is needed before assigning specific stabilizing interfaces within the lattice.
- 2.Analyze particle heterogeneity by cryo-ETassay method and structural subject
Assess heterogeneity in immature particle lattice curvature and CA-to-membrane spacing.
The abstract describes this as further analysis after the high-resolution reconstruction, adding particle-level heterogeneity information complementary to the reconstructed lattice model.
Objective: Enable in situ structural studies of the near-native cellular interior and surroundings by combining cryogenic sample preparation, lamella fabrication, tomography, and downstream data processing.
Why it works: The review abstract states that isolated-molecule cryo-EM can lose cellular-context information, whereas cryogenic FIB fabrication creates thin lamellae that permit cryo-ET access to the near-native cellular interior. The workflow therefore combines preservation, thinning, imaging, and analysis to recover in situ structural information.
thin lamella generation from cells and tissuestomographic imaging of cellular interiorspreservation of native molecular interactionsultrarapid sample freezingfocused ion beam lamella fabricationcryo-electron tomographydata processingcorrelative light and electron microscopy
Stages
- 1.Ultrarapid sample freezing(library_build)
The abstract names ultrarapid sample freezing as an enabling technological development for cryo-ET studies on FIB-generated lamellae.
Selection: Prepare samples under cryogenic conditions for downstream lamella fabrication and cryo-ET.
- 2.FIB fabrication of lamellae(library_build)
This stage exists because cryogenic FIB fabrication generates thin lamellae that enable cryo-ET structural studies of the near-native cellular interior and surroundings.
Selection: Generate thin lamellae from cellular samples and tissues for cryo-ET.
- 3.Cryo-electron tomography(functional_characterization)
Tomography is the imaging stage that provides structural information in the cellular environment after lamella preparation.
Selection: Acquire structural information from the near-native cellular interior and surroundings.
- 4.Data processing(secondary_characterization)
The abstract states that cellular cryo-ET benefits from data-processing developments and that more in situ structures are being obtained at increasingly higher resolution.
Selection: Process cryo-ET data to support structure determination and higher-resolution in situ analysis.
- 5.Correlative light and electron microscopy support(secondary_characterization)
The abstract names correlative light and electron microscopy as an enabling technological development for these studies.
Selection: Provide correlative imaging support within the cryo-ET on FIB-lamella workflow.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
correlative light and electron microscopycryogenic preservationelectron tomographyfocused ion beam lamella thinningTarget processes
recombinationInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensor
The method requires cryo-electron microscopy/tomography instrumentation and immature virus particle samples suitable for structural imaging.; requires cryo-EM/ET instrumentation and structural analysis workflow; The review states that cryo-ET on these samples depends on FIB-generated thin lamellae and benefits from electron microscopes, detectors, and data processing. Correlative light and electron microscopy is also named as an enabling development.; depends on cryogenic focused ion beam fabrication of thin lamellae for many cellular and tissue samples; relies on electron microscopes, detectors, and data processing
The abstract does not claim that cryo-ET alone removes all sample-preparation or resolution limitations. It still depends on upstream lamella fabrication and supporting instrumentation.; requires thin lamella preparation for cellular samples and tissues
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Cryogenic electron microscopy of isolated macromolecules can achieve near-atomic resolution but does not provide structural information in the cellular environment where macromolecules perform native functions.
Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions
Cryogenic focused ion beam fabrication generates thin lamellae from cellular samples and tissues, enabling cryo-electron tomography studies of the near-native cellular interior and surroundings.
Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).
Cellular cryo-ET benefits from developments in electron microscopes, detectors, and data processing, and more in situ structures are being obtained at increasingly higher resolution.
Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution.
Ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing, and correlative light and electron microscopy are enabling developments for cryo-ET studies on FIB-generated lamellae.
the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies
Approval Evidence
2 sources5 linked approval claimsfirst-pass slugs cryo-electron-microscopy-and-tomography, cryo-electron-tomography
Here, we characterize the immature CA lattice from immature virus particles by using cryo-electron microscopy and tomography (cryo-EM/ET).
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Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).
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morphology observationsupports
Cryo-ET revealed heterogeneity in HTLV-1 immature particles, including varying lattice curvatures and varying distances from the CA layer to the membrane.
Further analysis by cryo-ET reveals clear heterogeneity, notably the varying lattice curvatures and the varying distances from the CA layer to the membrane.
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comparative scopesupports
Cryogenic electron microscopy of isolated macromolecules can achieve near-atomic resolution but does not provide structural information in the cellular environment where macromolecules perform native functions.
Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions
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enabling rolesupports
Cryogenic focused ion beam fabrication generates thin lamellae from cellular samples and tissues, enabling cryo-electron tomography studies of the near-native cellular interior and surroundings.
Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).
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technology trendsupports
Cellular cryo-ET benefits from developments in electron microscopes, detectors, and data processing, and more in situ structures are being obtained at increasingly higher resolution.
Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution.
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workflow enablersupports
Ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing, and correlative light and electron microscopy are enabling developments for cryo-ET studies on FIB-generated lamellae.
the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies
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Comparisons
Source-stated alternatives
The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
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The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
Source-backed strengths
supports analysis of lattice heterogeneity in immature particles; provides structural information in the native cellular environment; benefits from improvements in microscopes, detectors, and data processing; increasingly yields higher-resolution in situ structures
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supports analysis of lattice heterogeneity in immature particles
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provides structural information in the native cellular environment
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benefits from improvements in microscopes, detectors, and data processing
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increasingly yields higher-resolution in situ structures
Compared with electron microscopy
The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
Shared frame: source-stated alternative in extracted literature
Strengths here: supports analysis of lattice heterogeneity in immature particles; provides structural information in the native cellular environment; benefits from improvements in microscopes, detectors, and data processing.
Relative tradeoffs: requires thin lamella preparation for cellular samples and tissues.
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The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
Compared with microscopy
The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
Shared frame: source-stated alternative in extracted literature
Strengths here: supports analysis of lattice heterogeneity in immature particles; provides structural information in the native cellular environment; benefits from improvements in microscopes, detectors, and data processing.
Relative tradeoffs: requires thin lamella preparation for cellular samples and tissues.
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The abstract contrasts cryo-ET with cryogenic electron microscopy of isolated macromolecules, which can reach near-atomic resolution but lacks cellular-context information.
Ranked Citations
- 1.