Source 1primary paper2011Nature Methods
Toolkit/4Pi detection of stochastically switched fluorophores
4Pi detection of stochastically switched fluorophores
Also known as: 4Pi setup
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores.
Usefulness & Problems
Why this is useful
This method performs 3D super-resolution imaging of stochastically switched fluorophores across whole cells using 4Pi detection. It localizes single markers in three dimensions and supports multicolor nanoscopy by polarization splitting.; 3D super-resolution imaging across whole cells; multicolor 3D nanoscopy; high-precision single-marker localization in three dimensions
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This method performs 3D super-resolution imaging of stochastically switched fluorophores across whole cells using 4Pi detection. It localizes single markers in three dimensions and supports multicolor nanoscopy by polarization splitting.
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3D super-resolution imaging across whole cells
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multicolor 3D nanoscopy
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high-precision single-marker localization in three dimensions
Problem solved
It addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.; extends axial imaging depth while maintaining nanoscale 3D localization; enables two-color or multicolor 3D nanoscopy in transparent samples
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It addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.
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extends axial imaging depth while maintaining nanoscale 3D localization
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enables two-color or multicolor 3D nanoscopy in transparent samples
Problem links
enables two-color or multicolor 3D nanoscopy in transparent samples
LiteratureIt addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.
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It addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.
extends axial imaging depth while maintaining nanoscale 3D localization
LiteratureIt addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.
Source:
It addresses the challenge of combining nanoscale 3D resolution, multicolor recording, and extended axial depth in noninvasive imaging of cells and other transparent materials.
Published Workflows
Objective: To achieve noninvasive 3D super-resolution imaging of stochastically switched fluorophores across whole cells with multicolor capability and extended axial depth.
Why it works: The abstract attributes improved 3D localization to evaluating higher moments of the diffraction spot in a 4Pi detection scheme, and attributes multicolor capability to splitting fluorescence into orthogonal polarization states.
4Pi detectionevaluation of higher moments of the diffraction spotorthogonal polarization splitting of fluorescence lightsingle-marker localization3D super-resolution imagingmulticolor nanoscopy
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
4pi interferometric detectionhigher-moment analysis of the diffraction spotpolarization-based signal separationstochastic fluorophore switchingTechniques
Functional AssayTarget processes
localizationInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensor
The abstract indicates that the method requires a 4Pi detection setup, stochastically switched fluorophores, and orthogonal-polarization splitting for multicolor operation.; requires a 4Pi detection scheme; requires stochastically switched fluorophores; multicolor implementation requires splitting fluorescence into orthogonal polarization states
The abstract does not establish performance in opaque samples or provide evidence about applications beyond transparent materials.; abstract only supports use in cells and other transparent materials
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The method advances noninvasive 3D imaging of cells and other transparent materials by combining multicolor recording, nanoscale resolution, and extended axial depth.
Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.
A 4Pi detection scheme applied to stochastically switched fluorophores enables 3D super-resolution imaging across whole cells.
We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells.
Splitting fluorescence light into orthogonal polarization states enables the 4Pi setup to perform 3D nanoscopy of multiple fluorophores.
By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores.
Evaluating higher moments of the diffraction spot in a 4Pi detection scheme allows simultaneous 3D localization of single markers with less than 10 nm precision within a 650 nm thick layer at an arbitrarily selected sample depth.
By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample.
3D localization precision 10 nmaxial layer thickness 650 nm
Approval Evidence
1 source4 linked approval claimsfirst-pass slug 4pi-detection-of-stochastically-switched-fluorophores
By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores.
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advancesupports
The method advances noninvasive 3D imaging of cells and other transparent materials by combining multicolor recording, nanoscale resolution, and extended axial depth.
Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.
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capabilitysupports
A 4Pi detection scheme applied to stochastically switched fluorophores enables 3D super-resolution imaging across whole cells.
We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells.
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capabilitysupports
Splitting fluorescence light into orthogonal polarization states enables the 4Pi setup to perform 3D nanoscopy of multiple fluorophores.
By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores.
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performancesupports
Evaluating higher moments of the diffraction spot in a 4Pi detection scheme allows simultaneous 3D localization of single markers with less than 10 nm precision within a 650 nm thick layer at an arbitrarily selected sample depth.
By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample.
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Comparisons
Source-stated alternatives
The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
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The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
Source-backed strengths
<10 nm 3D localization precision; 650 nm thick imaging layer at arbitrarily selected depth; supports multiple fluorophores via orthogonal polarization splitting
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<10 nm 3D localization precision
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650 nm thick imaging layer at arbitrarily selected depth
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supports multiple fluorophores via orthogonal polarization splitting
Compared with imaging
The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
Shared frame: source-stated alternative in extracted literature
Strengths here: <10 nm 3D localization precision; 650 nm thick imaging layer at arbitrarily selected depth; supports multiple fluorophores via orthogonal polarization splitting.
Relative tradeoffs: abstract only supports use in cells and other transparent materials.
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The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
Compared with imaging surveillance
The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
Shared frame: source-stated alternative in extracted literature
Strengths here: <10 nm 3D localization precision; 650 nm thick imaging layer at arbitrarily selected depth; supports multiple fluorophores via orthogonal polarization splitting.
Relative tradeoffs: abstract only supports use in cells and other transparent materials.
Source:
The abstract does not explicitly compare this setup against a named alternative method, though it positions the approach as an advance over prior 3D imaging capabilities.
Ranked Citations
- 1.