Correcting confocal acquisition to optimize imaging of fluorescence resonance energy by

sensitized emission

Jacco van Rheenen Kees Jalink

outline

Confocal FRET

IntroductionMajor sources of error and variability in confocal acquisitionSolutionsExamples

Fluorescence Resonance Energy Transfer

Energy Transfer

CFP YFP

•Efficiency (E): (#quanta transferred)/(#quanta absorbed):

E=R06/(R6+ R0

6) (Förster eq.)

FRET measurements

FRET and E

spatial and temporal

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

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

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

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)

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)

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

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

van Rheenen et al., 2004, Biophys. J. 86: 2517-29

Computer saves us lots of time

Computer saves us lots of time

Confocal vs Wide-field

ConfocalWide-field

Mercury lamp 2 laser lines

Single detector Two PMT

Axial resolutionNo axial resolution

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)

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

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)

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

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

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

PIP2 gradients?

CFP-PH Sens Ed

EPAC, a cAMP sensor

Ponsioen et al., EMBO reports 2004

Conclusion

Easy way to measure FRET

Great resolution

Cheap way to measure FRET

M. Langeslag B. PonsioenG. Van der KrogtK. Jalink

NWO grant 901-02-236