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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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chapter seven
XMUGXQ PFS0601Chapter Seven

Measurement of Fluorescence Lifetime

&

Time-domain Fluorescence

&

Frequency-domain Fluorescence

content
XMUGXQ PFS0601Content

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

7 1 introduction
XMUGXQ PFS06017.1 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

methods for measuring fluorescence lifetime
XMUGXQ PFS0601

I0

I0

t

t

Methods for measuring fluorescence lifetime
  • Pulse method

Light source

  • Harmonic or phase-modulation method

Light source

7 2 pulse lifetime measurement
XMUGXQ PFS0601

hvA

hvF

Log F(t)

or

log N(t)

S1

S1

knr

t

S0

7.2 pulse lifetime measurement
average lifetime
XMUGXQ PFS0601Average lifetime

1 ns, 1

2 ns, 1

3 ns, 1

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

multi component
XMUGXQ PFS0601

5 ns

50 ns

Multi-component

 Pre-exponential factor

7 3 phase and modulation measurements of fluorescence lifetime
XMUGXQ PFS06017.3 Phase and modulation measurements of fluorescence lifetime

Phase angle ()

Demodulation factor (m)

phase modulation method
XMUGXQ PFS0601Phase-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

phase modulation method2
XMUGXQ PFS0601Phase-modulation method

Single component

multicomponent

Average lifetime

7 4 measurement of time resolved decays of fluorescence
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

pulse sampling method
XMUGXQ PFS0601Pulse sampling Method

Photomultiplier

Multichannel analyzer MAC

single photon counting method
XMUGXQ PFS0601Single photon counting method

Time to amplitude converter TAC

Multichannel pulse heigh analyzer MCPHA

streak camera
XMUGXQ PFS0601Streak Camera

Simultaneous measurements of both wavelength and time resolved decays

analysis of time resolved decays of fluorescence intensity
XMUGXQ PFS0601

Log F(t)

or

log N(t)

t

Analysis of time-resolved decays of fluorescence intensity

In principle, for single exponential decay

analysis of time resolved decays of fluorescence intensity1
XMUGXQ PFS0601Analysis 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)

analysis of time resolved decays of fluorescence intensity2
XMUGXQ PFS0601Analysis 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

analysis of time resolved decays of fluorescence intensity3
XMUGXQ PFS0601Analysis of time-resolved decays of fluorescence intensity

Total intensity decay,

The purpose is to get F(t)

Commercial software available

analysis of time resolved decays of fluorescence intensity4
XMUGXQ PFS0601Analysis 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

analysis of time resolved decays of fluorescence intensity5
XMUGXQ PFS0601Analysis 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.

example
XMUGXQ PFS0601Example

Single exponential decay

Double exponential decay

example1
XMUGXQ PFS0601Example

Single exponential decay

Double exponential decay

7 5 application of time resolved fluorescence
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

example2
XMUGXQ PFS0601Example

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

application
XMUGXQ PFS0601

I

t

Application
  • Multi-components measurement
application1
XMUGXQ PFS0601

[Q]

Application
  • Dynamic quenching
application2
XMUGXQ PFS0601Application
  • Time-resolved fluorescence immunoassay

Example

TBP-Eu3+

XL665

D

A

TBP-Eu3+

链[霉]亲和素

example3
XMUGXQ PFS0601Example

Interference:

Background from media

Emission from free acceptor

application3
XMUGXQ PFS0601

background

Application

Free XL665

TBP-Eu3+(665nm/620nm)

FRET(665nm/620nm)

7 6 phase sensitive detection of fluorescence
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

principle
XMUGXQ PFS0601principle

For single component

Excited with

Emission

Phase sensitive detection (lock-in amplifer )

D, detection phase

principle1
XMUGXQ PFS0601

F

F

Principle

At i

At i

F change with cos(D-)

F change with 

principle2
XMUGXQ PFS0601principle

For two component

Emission

Phase sensitive detection

Phase depression

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