FRET Detection: Acceptor Photobleaching€¦ · molecular ruler in microscopy FRET is a...
Transcript of FRET Detection: Acceptor Photobleaching€¦ · molecular ruler in microscopy FRET is a...
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19.02.2013 1"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Detection: Acceptor PhotobleachingPrinciple of Acceptor Photobleaching
Acceptor photobleaching: a simple FRET detection method
De-quenching of the donor after selective photobleaching of the acceptor causes an increase in donor emission that can be readily quantified.
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19.02.2013 2"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
(1) Imaging Setup
• Multitrack Channel Mode Linewise switch (fast)
1) Excite Donor and DetectDonor emission
2) Excite Acceptor and DetectAcceptor emission
(2) Regions
• Select the cells / structures to be analyzed by defining bleachROIs (ROIs = Regions of Interest)
(3) Bleaching
• Acquire a couple of pre-bleach images to enable thesystem to calculate average intensities for analysis(baseline)
• Instead of one-shot bleach define repetetive bleach events(maximum delta donor intensity)
• Start with a pre-experiment to test bleaching conditions: „Stop when intensity drops to XX %“
• Apply high laser power / iteration• Bleach acceptor to ~ 10-20% of its initial value
(4) Time Series
• Acquire a number of time points
FRET Detection: Acceptor PhotobleachingZEN 2010: Image acquisition
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19.02.2013 3"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Detection: Acceptor PhotobleachingZEN 2010: Data evaluation using FRET
(1) FRET
• Method: Acceptor Bleach• Define ROIs: Enable
Object(s) and Background• NO reference possible
(2) Parameter
• Define acquisition andbleaching parameter
(3) Thresholds
• From image
1
2
3
Bleached ROI
Bleached ROI
Background
1
2
3
Bleached ROI
Bleached ROI
Background
(4) Data export
(1) (2) (3)
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19.02.2013 4"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET
sensitizedemission
Employing FRET (Fluorescence resonance energy transfer) as a molecular ruler in microscopy
FRET is a non-radiative transfer of an excited state from one fluorophore (donor) to another (acceptor).
FRET occurs if donor and acceptor are in close proximity (1-10 nm).
FRET permits microscopic proximity assays at the molecular level!
FRET Detection: Sensitized EmissionPrinciple of Sensitized Emission
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19.02.2013 5"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Detection: Sensitized EmissionZEN: Data evaluation in ZEN 2010 using FRET
(1) Imaging Setup
• Multitrack Channel Mode• Linewise switch (fast)
(1) Track1: Donor (blue) + FRET (yellow)(2) Track2: Acceptor (red)
(2) Acquire images of FRET sample and referenceswith the same settings
• Avoid noisy images (→Ratios)• Adjust settings for FRET sample• Acquire references with the same acquisition parameters
FRET
„Donor only“
„Acceptor only
Fd Ff Fa
Dd Df Da
Ad Af Aa
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19.02.2013 6"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
(1) Parameter Assign channels (→ Donor, → Acceptor, → FRET)(2) FRET Method Fc Youvan
Define ROIs / Enable Object(s) and Background(3) Thresholds „From image“(4) Parameter Analyse „Donor“
Save „Donor_Parameter.fret“
(A) Workflow „Donor only“
(1) (2) (3) (4)
FRET Detection: Sensitized EmissionZEN: Data evaluation in ZEN 2010 using FRET
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19.02.2013 7"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
(B) Workflow „Acceptor only“
(1) (2) (3) (4)
(1) Parameter Assign channels (→ Donor, → Acceptor, → FRET)(2) FRET Method Fc Youvan
Define ROIs / Enable Object(s) and Background(3) Thresholds „From image“(4) Parameter Analyse „Acceptor“
Save „Acceptor_Parameter.fret“
FRET Detection: Sensitized EmissionZEN: Data evaluation in ZEN 2010 using FRET
![Page 8: FRET Detection: Acceptor Photobleaching€¦ · molecular ruler in microscopy FRET is a non-radiative transfer of an excited state from one fluorophore (donor) to another (acceptor).](https://reader033.fdocuments.us/reader033/viewer/2022060603/60576c79a721491430278d7c/html5/thumbnails/8.jpg)
19.02.2013 8"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
(C) Workflow „FRET“
(1) (2) (3) (4)
(1) Parameter Assign channels (→ Donor, → Acceptor, → FRET)(2) FRET Method Fc Youvan
Define ROIs / Enable Object(s) and Background(3) Thresholds „From image“(4) Parameter Load „Donor“
Load „Acceptor“ (shown)
FRET Detection: Sensitized EmissionZEN: Data evaluation in ZEN 2010 using FRET
![Page 9: FRET Detection: Acceptor Photobleaching€¦ · molecular ruler in microscopy FRET is a non-radiative transfer of an excited state from one fluorophore (donor) to another (acceptor).](https://reader033.fdocuments.us/reader033/viewer/2022060603/60576c79a721491430278d7c/html5/thumbnails/9.jpg)
19.02.2013 9"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Detection: Sensitized EmissionQuantifying FRET: Sensitized Emission
Method 1
Fc (FRETcorrected)D.C. Youvan et al. 1997
This method calculates the absolute intensity of sensitized emission in the FRET channel as Fc. Therefore, high Fc values occur at high molecular concentrations.
... corrAcccorrDonorFfFc
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19.02.2013 10"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Microscopy with LSM SystemsQuantifying FRET: Sensitized Emission
Method 2
Fn (FRET net)G.W. Gordon et al. 1998
This method emphasizes FRET occurring at low concentrations of FRET pairs.
FaFdG
corrAcccorrDonorFfFn
...
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19.02.2013 11"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
FRET Microscopy with LSM SystemsQuantifying FRET: Sensitized Emission
Method 3
NF (normalized FRET)X. Xia et al. 2001
NF provides FRET values normalized for concentrations of
FRET pairs.
FaFdG
corrAcccorrDonorFfNF
...
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19.02.2013 12"Quick FRET Guide ZEN 2010" Training Application and Support Center - TASC
Sensitized Emission – The 3 (main) Methods
FaFdG
corrAcccorrDonorFfFn
...
... corrAcccorrDonorFfFc
FaFdG
corrAcccorrDonorFfNF
...
Method 1: Fc (FRET corrected) D.C. Youvan et al. 1997Dependend on Donor / Acceptor concentration. If its less → no FRET Signal
Method 2: Fn (FRET net) G.W. Gordon et al. 1998Facilitates getting FRET Signals also if Donor / Acceptor signals are weak
Method 3: NF (normalized FRET) X. Xia et al. 2001Independent of Donor und Akzeptor concentration