XMUGXQ PFS0601 Principles of Fluorescence Spectroscopy Chemistry Department XMU

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

XMUGXQ PFS 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

XMUGXQ PFS 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

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

XMUGXQ PFS pulse lifetime measurement S0S0 S1S1 S1S1 hv A hv F  k nr Log F(t) or log N(t) t

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

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

XMUGXQ PFS Phase and modulation measurements of fluorescence lifetime Phase angle (  ) Demodulation factor (m)

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

XMUGXQ PFS0601 Phase-modulation method

XMUGXQ PFS0601 Phase-modulation method Single component multicomponent Average lifetime

XMUGXQ PFS 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

XMUGXQ PFS0601 Pulse sampling Method Photomultiplier Multichannel analyzer MAC

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

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

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

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

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

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

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

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.

XMUGXQ PFS0601 Analysis of time-resolved decays of fluorescence intensity

XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

XMUGXQ PFS0601 Example Single exponential decay Double exponential decay

XMUGXQ PFS 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

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

XMUGXQ PFS0601 Application Multi-components measurement I t

XMUGXQ PFS0601 Application Dynamic quenching [Q][Q]

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

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

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

XMUGXQ PFS0601 Expression for the Figs

XMUGXQ PFS0601 Reference

XMUGXQ PFS0601 Time-resolved emission spectra

XMUGXQ PFS 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

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

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

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

XMUGXQ PFS0601 Phase sensitive detection fluorescence spectra

XMUGXQ PFS0601 Example

XMUGXQ PFS0601 Example

XMUGXQ PFS0601 Example

XMUGXQ PFS0601 Example