Source 1primary paper2014PLoS ONE
Toolkit/Dendra2 fused to actin
Dendra2 fused to actin
Also known as: Dendra2-actin
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments.
Usefulness & Problems
Why this is useful
Dendra2 fused to actin is used here as the photoconvertible fluorescent marker whose bleaching, blinking, and photoconversion yields are measured in cellulo during typical F-PALM experiments.; measuring in-cell phototransformation behavior of a photoconvertible fluorescent protein under F-PALM conditions; single-molecule localization microscopy marker characterization
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Dendra2 fused to actin is used here as the photoconvertible fluorescent marker whose bleaching, blinking, and photoconversion yields are measured in cellulo during typical F-PALM experiments.
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measuring in-cell phototransformation behavior of a photoconvertible fluorescent protein under F-PALM conditions
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single-molecule localization microscopy marker characterization
Problem solved
It enables direct measurement of phototransformation parameters in a biologically relevant cellular context needed for localization microscopy and molecular counting.; provides a biologically relevant in-cell fluorescent marker context for estimating photobleaching, blinking, and photoconversion yields
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It enables direct measurement of phototransformation parameters in a biologically relevant cellular context needed for localization microscopy and molecular counting.
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provides a biologically relevant in-cell fluorescent marker context for estimating photobleaching, blinking, and photoconversion yields
Problem links
provides a biologically relevant in-cell fluorescent marker context for estimating photobleaching, blinking, and photoconversion yields
LiteratureIt enables direct measurement of phototransformation parameters in a biologically relevant cellular context needed for localization microscopy and molecular counting.
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It enables direct measurement of phototransformation parameters in a biologically relevant cellular context needed for localization microscopy and molecular counting.
Published Workflows
Objective: Measure phototransformation quantum yields of Dendra2 in a biologically relevant in-cell context for typical F-PALM experiments.
Why it works: The abstract states that phototransformation parameters depend on local environment and therefore should be measured in cellulo in biologically relevant conditions; the workflow combines in-cell measurement, data processing, and simulations to obtain accurate yield estimates.
photoactivationphotoblinkingphotobleachinggreen-to-red photoconversionredox-based bleaching and blinking mechanismsF-PALMclustering optimization-based data processingsimulation-based parameter-range estimation
Stages
- 1.Simulation-based parameter estimation(in_silico_filter)
The abstract states that simulations were used to estimate the range of molecular density, molecular orientation, background level, laser power, and frametime adequate for accurate determination of phototransformation yields.
Selection: experimental parameter ranges adequate for accurate determination of phototransformation yields
- 2.In-cell phototransformation measurement(functional_characterization)
The abstract states that these parameters may depend on local environment and therefore should be measured in cellulo in biologically relevant experimental conditions.
Selection: measurement of photobleaching, blinking-off, blinking-on, and photoconversion parameters in fixed mammalian cells
- 3.Condition comparison with DABCO and noncellular reference(secondary_characterization)
The abstract reports that yields differed from those measured in PVA and were strongly affected by DABCO, indicating a comparative characterization stage to assess environmental effects on photophysics.
Selection: compare phototransformation yields across conditions including DABCO addition and PVA reference measurements
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
blinkingphotobleachingphotoconversionredox-based bleaching and blinkingthermal reactivationTechniques
No technique tags yet.
Target processes
recombinationInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: regulator
The abstract supports use in fixed mammalian cells under 561 nm or 405 nm illumination, with PBS buffer and optional DABCO affecting performance.; requires fixed mammalian cells; requires F-PALM-like illumination conditions; photophysical parameters depend on buffer/additives such as DABCO
The abstract does not show that this construct generalizes to all proteins, environments, or live-cell conditions.; reported measurements are specific to Dendra2 fused to actin in fixed mammalian cells and may depend on local environment
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successMammalian Cell Lineapplication demo
F-PALM
Inferred from claim c4 during normalization. DABCO improved Dendra2 photostability in fixed mammalian cells by reducing photobleaching yield about 2-fold, decreasing blinking-off yield more than 3-fold, and increasing blinking-on rate about 2-fold. Derived from claim c4.
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blinking-off yield fold change with DABCO(decreased more than 3-fold)blinking-on rate fold change with DABCO(increased 2-fold)photobleaching yield fold change with DABCO(reduced 2-fold)
Supporting Sources
Ranked Claims
DABCO improved Dendra2 photostability in fixed mammalian cells by reducing photobleaching yield about 2-fold, decreasing blinking-off yield more than 3-fold, and increasing blinking-on rate about 2-fold.
blinking-off yield fold change with DABCO decreased more than 3-foldblinking-on rate fold change with DABCO increased 2-foldphotobleaching yield fold change with DABCO reduced 2-fold
In fixed mammalian cells under typical F-PALM conditions, phototransformation quantum yields of Dendra2 fused to actin can be measured in cellulo.
The green-to-red photoconversion quantum yield of Dendra2 was estimated in cellulo under 405 nm illumination as 1.4 ± 0.6 × 10^-5.
green-to-red photoconversion quantum yield (1.4 ± 0.6) × 10(-5)
Under 561 nm illumination in PBS at pH 7.4, the blinking-off yield of Dendra2 fused to actin in fixed mammalian cells was measured as 2.3 ± 0.2 × 10^-5 and the thermally activated blinking-on rate as 11.7 ± 0.5 s^-1.
blinking-off yield (2.3 ± 0.2) × 10(-5)thermally-activated blinking-on rate 11.7 ± 0.5 s-1
Under 561 nm illumination in PBS at pH 7.4, the photobleaching yield of Dendra2 fused to actin in fixed mammalian cells was measured as 2.5 ± 0.4 × 10^-5.
photobleaching yield (2.5 ± 0.4) × 10(-5)
The observed Dendra2 bleaching and blinking behavior under F-PALM conditions is consistent with redox-based bleaching and blinking mechanisms.
A data processing strategy based on clustering optimization, together with simulations, was used to estimate phototransformation yields and define experimental parameter ranges adequate for accurate determination.
Approval Evidence
1 source6 linked approval claimsfirst-pass slug dendra2-fused-to-actin
Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments.
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comparative effectsupports
DABCO improved Dendra2 photostability in fixed mammalian cells by reducing photobleaching yield about 2-fold, decreasing blinking-off yield more than 3-fold, and increasing blinking-on rate about 2-fold.
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measurement resultsupports
In fixed mammalian cells under typical F-PALM conditions, phototransformation quantum yields of Dendra2 fused to actin can be measured in cellulo.
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measurement resultsupports
The green-to-red photoconversion quantum yield of Dendra2 was estimated in cellulo under 405 nm illumination as 1.4 ± 0.6 × 10^-5.
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measurement resultsupports
Under 561 nm illumination in PBS at pH 7.4, the blinking-off yield of Dendra2 fused to actin in fixed mammalian cells was measured as 2.3 ± 0.2 × 10^-5 and the thermally activated blinking-on rate as 11.7 ± 0.5 s^-1.
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measurement resultsupports
Under 561 nm illumination in PBS at pH 7.4, the photobleaching yield of Dendra2 fused to actin in fixed mammalian cells was measured as 2.5 ± 0.4 × 10^-5.
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mechanistic interpretationsupports
The observed Dendra2 bleaching and blinking behavior under F-PALM conditions is consistent with redox-based bleaching and blinking mechanisms.
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Comparisons
Source-stated alternatives
The abstract contrasts these in-cell measurements with measurements in poly-vinyl alcohol (PVA), but does not name an alternative fluorescent protein within the anchor paper abstract.
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The abstract contrasts these in-cell measurements with measurements in poly-vinyl alcohol (PVA), but does not name an alternative fluorescent protein within the anchor paper abstract.
Source-backed strengths
measured in fixed mammalian cells rather than only noncellular matrices; supports estimation of multiple phototransformation parameters relevant to molecular counting
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measured in fixed mammalian cells rather than only noncellular matrices
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supports estimation of multiple phototransformation parameters relevant to molecular counting
Dendra2 fused to actin and modular light-controlled skeletal muscle-powered bioactuator address a similar problem space because they share recombination.
Shared frame: same top-level item type; shared target processes: recombination; same primary input modality: light
Compared with Opto-Casp8-V2
Dendra2 fused to actin and Opto-Casp8-V2 address a similar problem space because they share recombination.
Shared frame: same top-level item type; shared target processes: recombination; same primary input modality: light
Compared with pcVP16
Dendra2 fused to actin and pcVP16 address a similar problem space because they share recombination.
Shared frame: same top-level item type; shared target processes: recombination; same primary input modality: light
Ranked Citations
- 1.