1. Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different materials Joel Hagman Supervisors Anne-Marie Hermansson Niklas Lorén

2. Outline Principles of CLSM Principles of FRAP Brief History of FRAP Bleaching modes Important parameters for FRAP FRAP in different materials Conclusions

3. Principles of Confocal Laser Scanning Microscopy (CLSM)

4. Principles of FRAP Braeckmans PhD thesis

5. Brief history of FRAP 1974 - Peters et al. 1976 - Axelrod et al. 1978 - Smith & McConnel 1982 - Davoust et al. 1983 - Soumpasis […] 1993 - Blonk et al. 1997 - Tinland et al. 2003 - Braeckmans et al. 2007 - Braeckmans et al. Features First attempt Stationary beam First tries of fringe pattern Enhanced fringe pattern Theory enhancement […] FRAP in CLSM FRAP & electrophoresis Bleached disc Bleached line Time

6. Bleaching modes Bleached diskBleached line Stationary beamFringe pattern Huyck

7. Stationary beam Axelrod, 1976 2D diffusion Easy Does not account for homo/heterogenety “Far from correct”

8. Stationary beam Soumpasis 1983

9. Fringe pattern Davoust, 1982 2D diffusion “Solves some issues from stationary beam” –Removes the dependency of the beam width –May allow a minor flow More complex equipment needed None CLSM technique

10. Fringe pattern Tinland 2000

11. Bleached disk Braeckmans, 2003 2D/3D diffusion Eliminates beam edge effects Requires a large bleach area Sensitive for heterogeneity Braeckmans 2006

12. Bleached disk Huyck Braeckmans 2003

13. Bleached line Braeckmans, 2007 1D diffusion Less accurate then disk model Can be performed in more heterogene areas Faster then disk model

14. Bleached line Huyck Braeckmans 2007

15. Important parameters for FRAP Effect of inter-line distance (Δy) ω = radial Gaussian resolution N = l/Δy+1 number of scans l = 20 w Braeckmans PhD thesis

16. Important parameters for FRAP Blonk 1993 Effect of Numerical Aperture (NA) Braeckmans PhD thesis Effect of bleach intensity distribution

17. FRAP in different materials Gels (some material) Solutions (a lot of material) Cells (a lot of material) Emulsions (none) Lipids (plenty of material) Gel electrophoresis (some material)

18. Gels Probe molecular weight (Stokes radius) combined with gelconcentration and dynamic viscosity Dynamic viscosity Mw decreases de Smedt 1997 Mw decreases

19. Gels Enzymatic treatment Burke 2000 β-mannanase creates backbone cleaving α-galactosidase creates side chain cleaving Enzyme modification time (min)

20. Solutions Burke 2000 Mw Bu 1994 Enzymatic treatmentMw and gel concentration Increasing gel concentration

21. Cells “FRAP revealed rapid dynamics of actin in spines” Star 2002

22. Cells Nuclear dynamics of AML1 fusion proteins in HeLa cells Qiu 2006

23. During electrophoresis Effect of electrical field & gel strength Ekani-Nkodo 2002

24. Fatty acid diffusion in heterogeneous chocolate determined using FRAP Directly after bleaching Before bleaching 10 s after bleaching 100 s after bleaching Bleached area Fatty acid analogue with Mw = 320 Da

25. Diffusion rate evaluated using Braeckmans uniform disk model 3 samples Fatty acid diffusion in heterogeneous chocolate determined using FRAP

26. Conclusions Possible to measure slow diffusion Versatile tool The technique have still a lot of growth potential The technique is “again” under development

27. Thank you for your attention Questions?