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 Energy Transfer CFPYFP Efficiency (E): (#quanta transferred)/(#quanta absorbed): E=R 0 6 /(R 6 + R 0 6 ) (Förster eq.)

4. FRET measurements FRET and E spatial and temporal

5. Energy Transfer Leakthrough complicates sensitized emission measurements Sensitized emission = acceptor fluorescence resulting from energy transfer from excited donor molecules CFPYFP Donor: CFP, excitation 430 nm Acceptor: YFP, excitation 514 nm Indirect excitation YFP Leak-through Donor CFP

6. PH CFP 430480530580630 CFP emission in Indirect YFP channel CFP 2 YFP PH Energy Transfer 430 nm  =CFP (in Indirect YFP Channel) CFP (in CFP Channel) YFP

7. CFP emission in Indirect YFP channel 430480530580630 CFP PH CFP 2 YFP PH Energy Transfer 430 nm  =CFP (in Indirect YFP Channel) CFP (in CFP Channel) YFP

8. 430480530580630 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)

9. 514 nm 430480530580630 Sensitized emission YFP PH CFP 2 YFP PH  =YFP (by 430 nm excitation source) YFP (by 514 nm excitation source)

10. 430 nm / Donor Excitation 430 nm / Donor 514 nm / Acceptor Emission collection CFP YFP M Donor M IndAcc M DirAcc Calculating Sensitized emission F sen =(M IndAcc -M Donor  -M DirAcc (  )/(1-  ) Gordon et al., 1998, Nagy et al., 1998, Hoppe et al., 2002 and van Rheenen et al., 2004

11. Sensitized emission and FRET efficiency 1000 CFP 1000 YFP 8 nm so 5%, 50 Sens 10 CFP 10 YFP 5 nm so 50%, 5 Sens 5 Sens / 10 CFP = 0.550 Sens / 1000 CFP = 0.05

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

13. Computer saves us lots of time

15. Confocal vs Wide-field ConfocalWide-field Mercury lamp 2 laser lines Single detectorTwo PMT Axial resolutionNo axial resolution

16. 1.31 Corr. 1.34 1.40 unCorr. 430 nm YFPSen 514 nm YFP unCorr. 0.93 Lateral spatial correction  = YFP (430 nm excitation) YFP (514 nm excitation)

17. Axial spatial correction 1 0 00.30.60.91.21.51.82.12.42.733.33.6 - Chromatic aberration

18. Direct YFP Iono +Ca CFP Sensitized YFP FRET efficiency -+ 0 0.5 0 2+ Laser fluctuation correction 2% 30 min 2% 30 min  = YFP (430 nm excitation) YFP (514 nm excitation)

19. Procedure 1. Donor excitation, collection Donor (M Donor ) and Acceptor M IndAcc 2. Refocusing preparation to minimize chromatic aberration effects 3. Acceptor excitation, collection Acceptor (M IndAcc ) 4. Shade correction 5. Calculating correction factors 6. Calculation sensitized emission, E D and E A

20. Procedure 1. Donor excitation, collection Donor (M Donor ) and Acceptor M IndAcc 2. Refocusing preparation to minimize chromatic aberration effects 3. Acceptor excitation, collection Acceptor (M IndAcc ) 4. Shade correction 5. Calculating correction factors 6. Calculation sensitized emission, E D and E A

22. CFP Membrane Cytosol PH PIP 2 2 2 2 YFP PH YFP PH CFP Energy Transfer Homogeneous FRET efficiency reveals homogeneous PIP 2 distribution CFP-PH SensEd

23. PIP 2 gradients? CFP-PH SensEd

24. EPAC, a cAMP sensor Ponsioen et al., EMBO reports 2004

25. Conclusion Easy way to measure FRET Great resolution Cheap way to measure FRET

26. M. Langeslag B. Ponsioen G. Van der Krogt K. Jalink NWO grant 901-02-236