correcting confocal acquisition to optimize imaging of fluorescence resonance energy by sensitized...
TRANSCRIPT
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Correcting confocal acquisition to optimize imaging of fluorescence resonance energy by
sensitized emission
Jacco van Rheenen Kees Jalink
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outline
Confocal FRET
IntroductionMajor sources of error and variability in confocal acquisitionSolutionsExamples
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Fluorescence Resonance Energy Transfer
Energy Transfer
CFP YFP
•Efficiency (E): (#quanta transferred)/(#quanta absorbed):
E=R06/(R6+ R0
6) (Förster eq.)
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FRET measurements
FRET and E
spatial and temporal
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Energy Transfer
Leakthrough complicates sensitized emission measurements
Sensitized emission = acceptor fluorescence resulting from energy transfer from excited donor molecules
CFP YFP Donor: CFP, excitation 430 nm
Acceptor: YFP, excitation 514 nm
Indirect excitation
YFP
Leak-through Donor
CFP
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PH
CFP
430 480 530 580 630
CFP emission in Indirect YFP channel
CFP
2
YFP
PH
Energy Transfer430 nm
=CFP (in Indirect YFP Channel) CFP (in CFP Channel)
YFP
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CFP emission in Indirect YFP channel
430 480 530 580 630
CFP
PH
CFP
2
YFP
PH
Energy Transfer430 nm
=CFP (in Indirect YFP Channel) CFP (in CFP Channel)
YFP
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430 480 530 580 630
Indirect YFP excitation
YFP
PH
CFP
2
YFP
PH
430 nm 514 nm
=YFP (by 430 nm excitation source) YFP (by 514 nm excitation source)
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514 nm
430 480 530 580 630
Sensitized emission
YFP
PH
CFP
2
YFP
PH
=YFP (by 430 nm excitation source) YFP (by 514 nm excitation source)
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430 nm / Donor
Excitation
430 nm / Donor
514 nm / Acceptor
Emission collection
CFP
YFP
YFP
MDonor
MIndAcc
MDirAcc
Calculating Sensitized emission
Fsen=(MIndAcc-MDonor-MDirAcc()/(1-)
Gordon et al., 1998, Nagy et al., 1998, Hoppe et al., 2002 and van Rheenen et al., 2004
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Sensitized emission and FRET efficiency
1000 CFP1000 YFP
8 nm so 5%, 50 Sens
10 CFP10 YFP
5 nm so 50%, 5 Sens
5 Sens / 10 CFP = 0.5 50 Sens / 1000 CFP = 0.05
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van Rheenen et al., 2004, Biophys. J. 86: 2517-29
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Computer saves us lots of time
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Computer saves us lots of time
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Confocal vs Wide-field
ConfocalWide-field
Mercury lamp 2 laser lines
Single detector Two PMT
Axial resolutionNo axial resolution
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1.31
Corr.Corr.
1.341.40
unCorr.
430 nmYFPSen
514 nmYFP
unCorr.
0.93
Lateral spatial correction
= YFP (430 nm excitation) YFP (514 nm excitation)
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Axial spatial correction
0
0.2
0.4
0.6
0.8
1
0 1 2 3
1
0
0 0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3 3.3
3.6 -
Chromatic aberration
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0 . 1
0 . 2
0 . 3
0 . 4
0 5 1 0 1 5 2 0
Ti m e ( m i n )
E
E online updated
E not updated
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
D
D
D
DirectYFP
Iono+Ca
CFP
Sensitized YFP
FRETefficiency
- +
0
0.5
0
0.5
2+
Laser fluctuation correction
2%
30 min
2%
30 min
= YFP (430 nm excitation) YFP (514 nm excitation)
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Procedure
1. Donor excitation, collection Donor (MDonor) and Acceptor MIndAcc
2. Refocusing preparation to minimize chromatic aberration effects
3. Acceptor excitation, collection Acceptor (MIndAcc)
4. Shade correction
5. Calculating correction factors
6. Calculation sensitized emission, ED and EA
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Procedure
1. Donor excitation, collection Donor (MDonor) and Acceptor MIndAcc
2. Refocusing preparation to minimize chromatic aberration effects
3. Acceptor excitation, collection Acceptor (MIndAcc)
4. Shade correction
5. Calculating correction factors
6. Calculation sensitized emission, ED and EA
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CFP
Membrane
Cytosol
PH
PIP2PIP2PIP2 PIP2
YFP
PH
YFP
PHPH
Membrane
Cytosol
PIP2 2PIP2PIP2 PIP2
YFP
PHPH
YFP
PHPH
CFP CFP CFP
Energy Transfer
Homogeneous FRET efficiency reveals homogeneous PIP2 distribution
CFP-PH Sens Ed
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PIP2 gradients?
CFP-PH Sens Ed
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EPAC, a cAMP sensor
Ponsioen et al., EMBO reports 2004
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Conclusion
Easy way to measure FRET
Great resolution
Cheap way to measure FRET
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M. Langeslag B. PonsioenG. Van der KrogtK. Jalink
NWO grant 901-02-236