Confocal microscopy vs Second harmonic generation microscopy.

1. Confocal microscopy vs Second harmonic generation microscopy. 光學系統期末報告 第B組 組員:馮培瑜 賴虢樺 羅尹駿 呂冠武 2012/01/03

2. 組員分工 A.馮培瑜 History Single photon and two photons excitation B.賴虢樺 Confocal Microscopy C.羅尹駿 Second Harmonic Generation (SHG) microscopy D.呂冠武 Compare PPT-maker

3. A • History • What is “fluorescence” • Single-photon fluorescence • Two-photonfluorescence

4. Microscope

5. Microscope=micro + skopos

6. History 13century, single type microscrope

7. History 13century, single type microscrope 16 century, compound microscope REF:http://info.edu.hc360.com/2006/07/25085795497.shtml

8. History 13century, single type microscrope 16 century, compound microscope 17 century, Hooke usedthe compound microscope REF:http://info.edu.hc360.com/2006/07/25085795497.shtml

9. History 13century, single type microscrope 16 century, compound microscope 17 century, Hooke use the compound microscope 1904, Köhler used the fluorescence microscopy to observe the sample. REF: http://www.bjsgyq.com/news/974.html

10. History 13century, single type microscrope 16 century, compound microscope 17 century, Hooke use the compound microscope 1904, Köhler use fluorescence microscopy to observe the sample. After 1970s, Confocal microscopy & Second harmonic generation microscrope come to the world. REF: http://pic.ypu.edu.tw/files/11-1029-1069.phphttp://www-ipcms.u-strasbg.fr/spip.php?article1146&lang=en

11. History 13century, single type microscrope 16 century, compound microscope 17 century, Hooke use the compound microscope 1904, Köhler use fluorescence microscopy to observe the sample. After 1970s, Confocal microscopy & Second harmonic generation microscrope come to the world. REF: http://pic.ypu.edu.tw/files/11-1029-1069.phphttp://www-ipcms.u-strasbg.fr/spip.php?article1146&lang=en

12. History 13century, single type microscrope 16 century, compound microscope 17 century, Hooke use the compound microscope 1904, Köhler use fluorescence microscopy to observe the sample. After 1970s, Confocal microscopy & Second harmonic generation microscrope come to the world. REF: http://pic.ypu.edu.tw/files/11-1029-1069.phphttp://www-ipcms.u-strasbg.fr/spip.php?article1146&lang=en

13. What is “fluorescence” REF:http://www.olympusmicro.com/primer/lightandcolor/fluorointroduction.html

14. Different kinds of fluorescence Auto-fluorescence (primary fluorescence) Secondary fluorescence

15. Single-photon fluorescence Use the single photonAr-UV laser ( about 351 nm ) to excite it, we will get the fluorescence with 461 nm wavelength. REF:雙光子吸收光致聚合技術應用於微元件製作之研究

16. Single-photon fluorescence Use the single photonAr-UV laser ( about 351 nm ) to excite it, we will get the fluorescence with 461 nm wavelength. UV light REF:雙光子吸收光致聚合技術應用於微元件製作之研究

17. Single-photon fluorescence Use the single photonAr-UV laser ( about 351 nm ) to excite it, we will get the fluorescence with 461 nm wavelength. UV light Cost REF:雙光子吸收光致聚合技術應用於微元件製作之研究

18. Two-photonfluorescence Use the double photonTi-Sapphire laser (702 nm) to excite it, we will get the same fluorescence with 461 nm wavelength. REF:雙光子吸收光致聚合技術應用於微元件製作之研究

19. Two-photonfluorescence Use the double photonTi-Sapphire laser (702 nm) to excite it, we will get the same fluorescence with 461 nm wavelength. lnfrared light REF:雙光子吸收光致聚合技術應用於微元件製作之研究

20. Two-photonfluorescence Use the double photonTi-Sapphire laser (702 nm) to excite it, we will get the same fluorescence with 461 nm wavelength. lnfrared light Replace UV light REF:雙光子吸收光致聚合技術應用於微元件製作之研究

21. B. • Confocal Microscopy • Fluorescence microscopy • Confocal Microscopy(part2) • Advantage and disadvantage • How to improve

22. 1.Confocal Microscopy • What is confocal In geometry, confocalmeans having the same focus.

23. 1.Confocal Microscopy • How does confocal microscopy work?

24. 2.Fluorescence microscopy

25. 2.Fluorescence microscopy • Nonconfocal

26. 3.Confocal microscopy(part2) Ref:http://www.olympusconfocal.com/theory/confocalintro.html

27. 3.Confocal microscopy(part2)

28. 3.Confocal microscopy(part2) • Compare with fluorescence microscopy

29. 4.Advantage and disadvantage • Advantage • Disadvantage

30. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage

31. Confocal microscopy is scanning point by point small pinhole  high resolution

32. Limit of resolutionand pinhole Airy disk d= 1.22 *λ *f/N For example illuminated light λ =600nm f/N=8 d=5.856μm  limit radius =5.856 μm

33. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage

34. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage a. Low efficiency

35. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage a. Low efficiency b. Photobleaching(光漂白)

36. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage a. Low efficiency b. Photobleaching(光漂白) c. Photodamage(光破壞) –heat up

37. 4.Advantage and disadvantage • Advantage a. High resolution • Disadvantage a. Low efficiency b. Photobleaching (光漂白) c. Photodamage(光破壞) -heat up d. Axis resolution

39. 5.How to improve • How can we solve this problem ?

40. Multiphoton fluorescence microscope!

42. Probability of excitation in certain area a. cross section  small b. intensity  high c. pulse laser required • Low energy wasted • Protect undetected specimen • Low energy laser • Higher axis resolution • Deeper penetration

43. Biological tissue absorption  spectrum

44. C. Second Harmonic Generation (SHG) microscopy • What is SHG?? • What is special about SHG? • Second harmonic imaging microscopy (SHIM) • Conclusion

45. 1.What is SHG? • Polarization • Nonlinear optics

46. 1.What is SHG? • Polarization • Nonlinear optics The second term is called SHG

47. 2.What is special about SHG? Assume the incident light can be simply expressed as the form Plug it in the SHG term

48. 2.What is special about SHG? Assume the incident light can be simply expressed as the form Plug it in the SHG term We get a double frequency!!

49. 2.What is special about SHG? (cont’d) TPEF SHG E2 E1 E0 RealTransitions VirtualTransitions

50. 2.What is special about SHG? (cont’d) TPEF SHG E2 E1 Energy is conserved!! E0 RealTransitions VirtualTransitions