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Principles of Fluorescence Spectroscopy

Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Seven. Measurement of Fluorescence Lifetime & Time-domain Fluorescence & Frequency-domain Fluorescence. Content. 7.1 introduction 7.2 pulse lifetime measurement

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Principles of Fluorescence Spectroscopy

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  1. XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU

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

  3. XMUGXQ PFS0601 Content 7.1 introduction 7.2 pulse lifetime measurement 7.3 Phase and Modulation Measurements of Fluorescence Lifetime 7.4 Measurement of Time-resolved Decays of Fluorescence 7.5 Application of Time-resolved Fluorescence 7.6 Phase-sensitive Detection of Fluorescence 7.7 Application of PSDF

  4. 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. XMUGXQ PFS0601 I0 I0 t t Methods for measuring fluorescence lifetime • Pulse method Light source • Harmonic or phase-modulation method Light source

  6. XMUGXQ PFS0601 hvA hvF Log F(t) or log N(t) S1 S1  knr t S0 7.2 pulse lifetime measurement

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

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

  10. 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 miand i, calculate average lifetime

  11. XMUGXQ PFS0601 Phase-modulation method

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

  13. XMUGXQ PFS0601 I0 t 7.4 Measurement of Time-resolved Decays of Fluorescence Pulse lifetime measurement • Pulse width Enough shorter compare to decay of fluorescence ps, fs • Photon counts Enough for accurate measurement Repeat excite

  14. XMUGXQ PFS0601 Pulse sampling Method Photomultiplier Multichannel analyzer MAC

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

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

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

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

  19. 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 ti, a large number of pulses with equal width ti, each induce an impulse response in the sample t - ti, emission delay compare to excitation

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

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

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

  24. XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

  25. XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

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

  27. 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. XMUGXQ PFS0601 I t Application • Multi-components measurement

  29. XMUGXQ PFS0601 [Q] Application • Dynamic quenching

  30. XMUGXQ PFS0601 Application • Time-resolved fluorescence immunoassay Example TBP-Eu3+ XL665 D A TBP-Eu3+ 链[霉]亲和素

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

  32. XMUGXQ PFS0601 background Application Free XL665 TBP-Eu3+(665nm/620nm) FRET(665nm/620nm)

  33. XMUGXQ PFS0601 Expression for the Figs

  34. XMUGXQ PFS0601 Reference

  35. XMUGXQ PFS0601 Time-resolved emission spectra

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

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

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

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

  40. XMUGXQ PFS0601 Phase sensitive detection fluorescence spectra

  41. XMUGXQ PFS0601 Example

  42. XMUGXQ PFS0601 Example

  43. XMUGXQ PFS0601 Example

  44. XMUGXQ PFS0601 Example

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