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Correcting confocal acquisition to optimize imaging of fluorescence resonance energy by sensitized emission

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Title: Correcting confocal acquisition to optimize imaging of fluorescence resonance energy by sensitized emission


1
Correcting confocal acquisition to optimize
imaging of fluorescence resonance energy by
sensitized emission
Jacco van Rheenen Kees Jalink
2
outline
Confocal FRET Introduction Major sources of
error and variability in confocal
acquisition Solutions Examples
3
Fluorescence Resonance Energy Transfer
  • Efficiency (E) (quanta transferred)/(quanta
    absorbed)
  • ER06/(R6 R06) (Förster eq.)

4
FRET measurements
FRET and E
spatial and temporal
5
Leakthrough complicates sensitized emission
measurements
Sensitized emission acceptor fluorescence
resulting from energy transfer from
excited donor molecules
Energy Transfer
CFP
YFP
6
CFP emission in Indirect YFP channel
PH
430 nm
430
480
530
580
630
CFP
YFP
7
CFP emission in Indirect YFP channel
2
PH
PH
YFP
Energy Transfer
430 nm
430
480
530
580
630
CFP
YFP
8
Indirect YFP excitation
2
PH
PH
YFP
430 nm
430
480
530
580
630
YFP
9
Sensitized emission
2
PH
PH
YFP
514 nm
430
480
530
580
630
YFP
10
Calculating Sensitized emission
Excitation
Emission collection
430 nm / Donor
CFP
MDonor
430 nm / Donor
YFP
MIndAcc
514 nm / Acceptor
YFP
MDirAcc
11
Sensitized emission and FRET efficiency
1000 CFP
10 CFP
1000 YFP
10 YFP
8 nm so 5, 50 Sens
5 nm so 50, 5 Sens
5 Sens / 10 CFP 0.5
50 Sens / 1000 CFP 0.05
12
van Rheenen et al., 2004, Biophys. J. 86 2517-29
13
Computer saves us lots of time
14
Computer saves us lots of time
15
Confocal vs Wide-field
Confocal
Wide-field
Mercury lamp
2 laser lines
Single detector
Two PMT
Axial resolution
No axial resolution
16
Lateral spatial correction
unCorr.
unCorr.
430 nm YFPSen
514 nm YFP
1.40
0.93
g YFP (430 nm excitation) YFP (514 nm
excitation)
17
Axial spatial correction
18
Laser fluctuation correction
g YFP (430 nm excitation) YFP (514 nm
excitation)
19
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
20
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
21
(No Transcript)
22
Homogeneous FRET efficiency reveals homogeneous
PIP2 distribution
Membrane
PIP
PIP
PIP
PIP
2
2
2
2
PH
PH
PH
PH
YFP
YFP
Cytosol
Energy Transfer
Sens
Ed
CFP-PH
23
PIP2 gradients?
Sens
Ed
CFP-PH
24
EPAC, a cAMP sensor
Ponsioen et al., EMBO reports 2004
25
Conclusion
Easy way to measure FRET
Cheap way to measure FRET
Great resolution
26
M. Langeslag B. Ponsioen G. Van der Krogt K.
Jalink
NWO grant 901-02-236
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