1. 20040400 XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU

2. 20040400 XMUGXQ PFS0601 Chapter Seven Measurement of Fluorescence Lifetime & Time-domain Fluorescence & Frequency-domain Fluorescence

3. 20040400 XMUGXQ PFS0601 7.1 introductionintroduction 7.2 pulse lifetime measurementpulse lifetime measurement 7.3 Phase and Modulation Measurements of Fluorescence LifetimePhase and Modulation Measurements of Fluorescence Lifetime 7.4 Measurement of Time-resolved Decays of FluorescenceMeasurement of Time-resolved Decays of Fluorescence 7.5 Application of Time-resolved FluorescenceApplication of Time-resolved Fluorescence 7.6 Phase-sensitive Detection of FluorescencePhase-sensitive Detection of Fluorescence 7.7 Application of PSDF Content

4. 20040400 XMUGXQ PFS0601 7.1 Introduction Information given by fluorescence lifetime The frequency of collisional encounters The rate of energy transfer The rate of excited state reaction Information related to its environment And so on

5. 20040400 XMUGXQ PFS0601 Methods for measuring fluorescence lifetime Pulse method Harmonic or phase-modulation method I0I0 t I0I0 t Light source

6. 20040400 XMUGXQ PFS0601 7.2 pulse lifetime measurement S0S0 S1S1 S1S1 hv A hv F  k nr Log F(t) or log N(t) t

7. 20040400 XMUGXQ PFS0601 Average lifetime 1 ns, 1 2 ns, 1 3 ns, 1 For a large number of fluorophores and small time interval, this sum becomes

8. 20040400 XMUGXQ PFS0601 Multi-component  Pre-exponential factor 5 ns 50 ns

9. 20040400 XMUGXQ PFS0601 7.3 Phase and modulation measurements of fluorescence lifetime Phase angle (  ) Demodulation factor (m)

10. 20040400 XMUGXQ PFS0601 Phase-modulation method  =2  f an important factor For small lifetime, set large modulation frequency For large lifetime, set small modulation frequency Choosing modulation frequency, let m = 0.3 ~ 0.7,  = 30 ~ 70º For commercial frequency-domain instrument, changing , measuring m i and  i, calculate average lifetime

11. 20040400 XMUGXQ PFS0601 Phase-modulation method

12. 20040400 XMUGXQ PFS0601 Phase-modulation method Single component multicomponent Average lifetime

13. 20040400 XMUGXQ PFS0601 7.4 Measurement of Time-resolved Decays of Fluorescence Pulse lifetime measurement Pulse width I0I0 t ps, fsEnough shorter compare to decay of fluorescence Photon counts Enough for accurate measurementRepeat excite

14. 20040400 XMUGXQ PFS0601 Pulse sampling Method Photomultiplier Multichannel analyzer MAC

15. 20040400 XMUGXQ PFS0601 Single photon counting method Time to amplitude converter TAC Multichannel pulse heigh analyzer MCPHA

16. 20040400 XMUGXQ PFS0601 Streak Camera Simultaneous measurements of both wavelength and time resolved decays

17. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity In principle, for single exponential decay Log F(t) or log N(t) t

18. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity Intensity profile of light, L(t) Intensity decay of fluorescence, F(t) Measured intensity decay, R(t) In practice, in consideration of the pulse width of lamp and multi-exponential decay

19. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity In practicing measurement Measuring the lamp profile, L(t), by using a solution which scatters light Measuring the total intensity decay, R(t), by using the sample At t i, a large number of pulses with equal width  t i, each induce an impulse response in the sample t - t i, emission delay compare to excitation

20. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity Total intensity decay, Commercial software available The purpose is to get F(t)

21. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity Least –squares analysis of time-resolved decays Let the number of components to be n, Give initial values to  i and  i, and calculate, get L(t), was measured

22. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity The  i and  i values are varied until the best fit is observed. A minimum value of  2 indicates the best fit. In this expression the sum extends over the number (n) of channels or data points used for a particular analysis.

23. 20040400 XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity

24. 20040400 XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

25. 20040400 XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

26. 20040400 XMUGXQ PFS0601 7.5 Application of time-resolved fluorescence Deduct back ground I t Light Back ground Target component Gated time Sampling time Measure intensity ex/ em Boxcar integrator Fast scan ex/ scan em

27. 20040400 XMUGXQ PFS0601 Example 荧光素标记荧光免疫分析，常常受到血液样品中胆红素背 景荧光干扰，采用时间分辨荧光免疫分析可以有效地消除 干扰。 荧光素寿命 3.6±0.46 ns, 测定时间 6.0 ns 胆红素寿命 0.21±0.14 ns, 测定时间 信号为零

28. 20040400 XMUGXQ PFS0601 Application Multi-components measurement I t

29. 20040400 XMUGXQ PFS0601 Application Dynamic quenching [Q][Q]

30. 20040400 XMUGXQ PFS0601 Application Time-resolved fluorescence immunoassay TBP-Eu 3+ XL665 链 [ 霉 ] 亲和素 Example TBP-Eu 3+ DA

31. 20040400 XMUGXQ PFS0601 Example Interference: Background from media Emission from free acceptor

32. 20040400 XMUGXQ PFS0601 Application background Free XL665 TBP-Eu 3+ (665nm/620nm) FRET(665nm/620nm)

33. 20040400 XMUGXQ PFS0601 Expression for the Figs

34. 20040400 XMUGXQ PFS0601 Reference

35. 20040400 XMUGXQ PFS0601 Time-resolved emission spectra

36. 20040400 XMUGXQ PFS0601 7.6 Phase-sensitive detection of fluorescence Principle F(t) I(t) F(t) m L =b/a m A =B/A m B =B’/A’ m L >m A >m B A<BA<B A<BA<B

37. 20040400 XMUGXQ PFS0601 principle For single component Excited with Emission Phase sensitive detection (lock-in amplifer )  D, detection phase

38. 20040400 XMUGXQ PFS0601 Principle At i F  F change with cos(  D -  ) At  i F change with F

39. 20040400 XMUGXQ PFS0601 principle For two component Emission Phase sensitive detection Phase depression

40. 20040400 XMUGXQ PFS0601 Phase sensitive detection fluorescence spectra

41. 20040400 XMUGXQ PFS0601 Example

42. 20040400 XMUGXQ PFS0601 Example

43. 20040400 XMUGXQ PFS0601 Example

44. 20040400 XMUGXQ PFS0601 Example