Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different materials

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. 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 Brief history of FRAP Time

6. Bleaching modes Stationary beam Fringe pattern Huyck Huyck Bleached disk Bleached line

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 Effect of Numerical Aperture (NA) Effect of bleach intensity distribution Braeckmans PhD thesis Blonk 1993

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 Mw decreases Mw decreases Dynamic viscosity de Smedt 1997

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

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

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 Bleached area Before bleaching Directly after bleaching 10 s after bleaching 100 s after bleaching Fatty acid analogue with Mw = 320 Da

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

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?