Title: Correcting confocal acquisition to optimize imaging of fluorescence resonance energy by sensitized emission
1Correcting confocal acquisition to optimize
imaging of fluorescence resonance energy by
sensitized emission
Jacco van Rheenen Kees Jalink
2outline
Confocal FRET Introduction Major sources of
error and variability in confocal
acquisition Solutions Examples
3Fluorescence Resonance Energy Transfer
- Efficiency (E) (quanta transferred)/(quanta
absorbed) - ER06/(R6 R06) (Förster eq.)
4FRET measurements
FRET and E
spatial and temporal
5Leakthrough complicates sensitized emission
measurements
Sensitized emission acceptor fluorescence
resulting from energy transfer from
excited donor molecules
Energy Transfer
CFP
YFP
6CFP emission in Indirect YFP channel
PH
430 nm
430
480
530
580
630
CFP
YFP
7CFP emission in Indirect YFP channel
2
PH
PH
YFP
Energy Transfer
430 nm
430
480
530
580
630
CFP
YFP
8Indirect YFP excitation
2
PH
PH
YFP
430 nm
430
480
530
580
630
YFP
9Sensitized emission
2
PH
PH
YFP
514 nm
430
480
530
580
630
YFP
10Calculating Sensitized emission
Excitation
Emission collection
430 nm / Donor
CFP
MDonor
430 nm / Donor
YFP
MIndAcc
514 nm / Acceptor
YFP
MDirAcc
11Sensitized 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
12van Rheenen et al., 2004, Biophys. J. 86 2517-29
13Computer saves us lots of time
14Computer saves us lots of time
15Confocal vs Wide-field
Confocal
Wide-field
Mercury lamp
2 laser lines
Single detector
Two PMT
Axial resolution
No axial resolution
16Lateral spatial correction
unCorr.
unCorr.
430 nm YFPSen
514 nm YFP
1.40
0.93
g YFP (430 nm excitation) YFP (514 nm
excitation)
17Axial spatial correction
18Laser fluctuation correction
g YFP (430 nm excitation) YFP (514 nm
excitation)
19Procedure
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
20Procedure
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)
22Homogeneous 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
23PIP2 gradients?
Sens
Ed
CFP-PH
24EPAC, a cAMP sensor
Ponsioen et al., EMBO reports 2004
25Conclusion
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