Principles of fluorescence spectroscopy
This presentation is the property of its rightful owner.
Sponsored Links
1 / 44

Principles of Fluorescence Spectroscopy PowerPoint PPT Presentation


  • 127 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Principles of Fluorescence Spectroscopy

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Principles of fluorescence spectroscopy

XMUGXQ PFS0601

Principles of Fluorescence Spectroscopy

Chemistry Department

XMU


Chapter seven

XMUGXQ PFS0601

Chapter Seven

Measurement of Fluorescence Lifetime

&

Time-domain Fluorescence

&

Frequency-domain Fluorescence


Content

XMUGXQ PFS0601

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


7 1 introduction

XMUGXQ PFS0601

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


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 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


Multi component

XMUGXQ PFS0601

5 ns

50 ns

Multi-component

 Pre-exponential factor


7 3 phase and modulation measurements of fluorescence lifetime

XMUGXQ PFS0601

7.3 Phase and modulation measurements of fluorescence lifetime

Phase angle ()

Demodulation factor (m)


Phase modulation method

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 miand i, calculate average lifetime


Phase modulation method1

XMUGXQ PFS0601

Phase-modulation method


Phase modulation method2

XMUGXQ PFS0601

Phase-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 PFS0601

Pulse sampling Method

Photomultiplier

Multichannel analyzer MAC


Single photon counting method

XMUGXQ PFS0601

Single photon counting method

Time to amplitude converter TAC

Multichannel pulse heigh analyzer MCPHA


Streak camera

XMUGXQ PFS0601

Streak 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 PFS0601

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)


Analysis of time resolved decays of fluorescence intensity2

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 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 PFS0601

Analysis 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 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


Analysis of time resolved decays of fluorescence intensity5

XMUGXQ PFS0601

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.


Analysis of time resolved decays of fluorescence intensity6

XMUGXQ PFS0601

Analysis of time-resolved decays of fluorescence intensity


Example

XMUGXQ PFS0601

Example

Single exponential decay

Double exponential decay


Example1

XMUGXQ PFS0601

Example

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 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


Application

XMUGXQ PFS0601

I

t

Application

  • Multi-components measurement


Application1

XMUGXQ PFS0601

[Q]

Application

  • Dynamic quenching


Application2

XMUGXQ PFS0601

Application

  • Time-resolved fluorescence immunoassay

Example

TBP-Eu3+

XL665

D

A

TBP-Eu3+

链[霉]亲和素


Example3

XMUGXQ PFS0601

Example

Interference:

Background from media

Emission from free acceptor


Application3

XMUGXQ PFS0601

background

Application

Free XL665

TBP-Eu3+(665nm/620nm)

FRET(665nm/620nm)


Expression for the figs

XMUGXQ PFS0601

Expression for the Figs


Reference

XMUGXQ PFS0601

Reference


Time resolved emission spectra

XMUGXQ PFS0601

Time-resolved emission spectra


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 PFS0601

principle

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 PFS0601

principle

For two component

Emission

Phase sensitive detection

Phase depression


Phase sensitive detection fluorescence spectra

XMUGXQ PFS0601

Phase sensitive detection fluorescence spectra


Example4

XMUGXQ PFS0601

Example


Example5

XMUGXQ PFS0601

Example


Example6

XMUGXQ PFS0601

Example


Example7

XMUGXQ PFS0601

Example


  • Login