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## PowerPoint Slideshow about ' Principles of Fluorescence Spectroscopy' - lorin

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XMUGXQ PFS0601Chapter Seven

Measurement of Fluorescence Lifetime

&

Time-domain Fluorescence

&

Frequency-domain Fluorescence

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

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

XMUGXQ PFS0601

I0

I0

t

t

Methods for measuring fluorescence lifetime- Pulse method

Light source

- Harmonic or phase-modulation method

Light source

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

XMUGXQ PFS06017.3 Phase and modulation measurements of fluorescence lifetime

Phase angle ()

Demodulation factor (m)

XMUGXQ PFS0601Phase-modulation method

=2f 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

XMUGXQ PFS0601

I0

t

7.4 Measurement of Time-resolved Decays of FluorescencePulse lifetime measurement

- Pulse width

Enough shorter compare to decay of fluorescence

ps, fs

- Photon counts

Enough for accurate measurement

Repeat excite

XMUGXQ PFS0601Single photon counting method

Time to amplitude converter TAC

Multichannel pulse heigh analyzer MCPHA

XMUGXQ PFS0601

Log F(t)

or

log N(t)

t

Analysis of time-resolved decays of fluorescence intensityIn principle, for single exponential decay

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)

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

XMUGXQ PFS0601Analysis of time-resolved decays of fluorescence intensity

Total intensity decay,

The purpose is to get F(t)

Commercial software available

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

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.

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

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

XMUGXQ PFS0601Application

- Time-resolved fluorescence immunoassay

Example

TBP-Eu3+

XL665

D

A

TBP-Eu3+

链[霉]亲和素

XMUGXQ PFS0601

I(t)

mL=b/a

F(t)

mA=B/A

F(t)

mB=B’/A’

7.6 Phase-sensitive detection of fluorescencePrinciple

A<B

mL>mA>mB

A<B

XMUGXQ PFS0601principle

For single component

Excited with

Emission

Phase sensitive detection (lock-in amplifer )

D, detection phase

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