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Principles of Fluorescence Spectroscopy

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Principles of Fluorescence Spectroscopy

Chemistry Department

XMU

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Measurement of Fluorescence Lifetime

&

Time-domain Fluorescence

&

Frequency-domain Fluorescence

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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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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

- 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

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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

Pre-exponential factor

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Phase angle ()

Demodulation factor (m)

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=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

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Single component

multicomponent

Average lifetime

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I0

t

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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Photomultiplier

Multichannel analyzer MAC

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Time to amplitude converter TAC

Multichannel pulse heigh analyzer MCPHA

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Simultaneous measurements of both wavelength and time resolved decays

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Log F(t)

or

log N(t)

t

In principle, for single exponential decay

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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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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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Total intensity decay,

The purpose is to get F(t)

Commercial software available

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- 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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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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Single exponential decay

Double exponential decay

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Single exponential decay

Double exponential decay

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I

t

- 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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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

- Multi-components measurement

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[Q]

- Dynamic quenching

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- Time-resolved fluorescence immunoassay

Example

TBP-Eu3+

XL665

D

A

TBP-Eu3+

链[霉]亲和素

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Interference:

Background from media

Emission from free acceptor

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background

Free XL665

TBP-Eu3+(665nm/620nm)

FRET(665nm/620nm)

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I(t)

mL=b/a

F(t)

mA=B/A

F(t)

mB=B’/A’

Principle

A<B

mL>mA>mB

A<B

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For single component

Excited with

Emission

Phase sensitive detection (lock-in amplifer )

D, detection phase

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F

F

At i

At i

F change with cos(D-)

F change with

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For two component

Emission

Phase sensitive detection

Phase depression

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