Principles of fluorescence spectroscopy
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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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Principles of Fluorescence Spectroscopy

Chemistry Department

XMU


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Chapter Seven

Measurement of Fluorescence Lifetime

&

Time-domain Fluorescence

&

Frequency-domain Fluorescence


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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


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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


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I0

I0

t

t

Methods for measuring fluorescence lifetime

  • Pulse method

Light source

  • Harmonic or phase-modulation method

Light source


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hvA

hvF

Log F(t)

or

log N(t)

S1

S1

knr

t

S0

7.2 pulse lifetime measurement


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Average lifetime

1 ns, 1

2 ns, 1

3 ns, 1

For a large number of fluorophores and small time interval, this sum becomes


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5 ns

50 ns

Multi-component

 Pre-exponential factor


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7.3 Phase and modulation measurements of fluorescence lifetime

Phase angle ()

Demodulation factor (m)


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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


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Phase-modulation method


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Phase-modulation method

Single component

multicomponent

Average lifetime


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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


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Pulse sampling Method

Photomultiplier

Multichannel analyzer MAC


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Single photon counting method

Time to amplitude converter TAC

Multichannel pulse heigh analyzer MCPHA


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Streak Camera

Simultaneous measurements of both wavelength and time resolved decays


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Log F(t)

or

log N(t)

t

Analysis of time-resolved decays of fluorescence intensity

In principle, for single exponential decay


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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)


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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


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Analysis of time-resolved decays of fluorescence intensity

Total intensity decay,

The purpose is to get F(t)

Commercial software available


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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


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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.


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Analysis of time-resolved decays of fluorescence intensity


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Example

Single exponential decay

Double exponential decay


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Example

Single exponential decay

Double exponential decay


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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


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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


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I

t

Application

  • Multi-components measurement


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[Q]

Application

  • Dynamic quenching


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Application

  • Time-resolved fluorescence immunoassay

Example

TBP-Eu3+

XL665

D

A

TBP-Eu3+

链[霉]亲和素


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Example

Interference:

Background from media

Emission from free acceptor


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background

Application

Free XL665

TBP-Eu3+(665nm/620nm)

FRET(665nm/620nm)


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Expression for the Figs


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Reference


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Time-resolved emission spectra


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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


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principle

For single component

Excited with

Emission

Phase sensitive detection (lock-in amplifer )

D, detection phase


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F

F

Principle

At i

At i

F change with cos(D-)

F change with 


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principle

For two component

Emission

Phase sensitive detection

Phase depression


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Phase sensitive detection fluorescence spectra


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Example


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Example


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Example


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Example


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