20040200 XMUGXQ PFS01-ITF01 Principles of Fluorescence Spectroscopy Chemistry Department XMU

20040200

XMUGXQ PFS01-ITF01

Principles of Fluorescence Spectroscopy

Chemistry DepartmentXMU

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Reference books 陈国珍等人编 , 荧光分析法，第二版，科学出版

社， 1990

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1999 (Ed.2) 1983 (Ed.1)

J. R. Lakowicz ed. Topics in Fluorescence Spectroscopy, 1-5, Plenum Press, New York

陈国珍等人编，荧光分析进展，厦门大学出版社，1992

B. Herman, Fluorescence Microscopy 2 ed. University of North Carolina

Research papers

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Course Requirements: One term paper, reviews including relevant r

esearch papers from 1998 to 2003. Self-chosen topic. 20%

One oral report (same topic as term paper)

（ 12 mins report, 3 mins defence) 20%

One translation (From Science, related to luminescence) 10%

Send to [email protected]

One test (basic principles) 50%

mailto:[email protected]

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关于教学

• 教学方式：讲授与研修、论文报告

• 评价：教师评价与学生互评结合

• 总课时： 54

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INTRODUCTION TO

FLUORESCENCE

Chapter One

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Introduction to Fluorescence

• 1.0 Introduction• 1.1 Phenomenon of Fluorescence• 1.2 Excitation and deactivation of molecule• 1.3 Models of molecular emission• 1.4 Characteristics of fluorescence emission• 1.5 Characteristics of fluorophore• 1.6 Time scale of Fluorescence• 1.7 Intensity and Concentration• 1.8 Fluorophore

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Fluorimetry Spectrofluorimetry

Molecular Fluorescence

Atomic Fluorescence

X Ray Fluorescence

Modern fluorescence spectroscopy

Fluorescence immunoassay

Fluorescence microscopy Confocal Fluorescence microscopy

Multiphoton microscopy fluorescence

Fluorescence lifetime image

Fluorescence polarization immunoassay

Time resolved fluorescenceTime-domain fluorescence

Frequency-domain fluorescence

XQGuo

x射线光子与原子发生碰撞时，驱逐一个内层电子而出现一个空穴，使整个原子体系处于不稳定的激发态。激发态的原子寿命为10-12~10-14s。俄歇效应：当较外层电子跃入空穴时，所释放的能量随即在原子内部被吸收而逐出较外层的另一个次级光电子，成为俄歇电子。x射线荧光：当较外层电子跃入内层空穴所释放的能量不在原子内被吸收，而以辐射形式释放，产生x射线荧光。

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Immunoassay

Environmental monitoring

Clinical chemistry

DNA sequencing

Genetic analysis

(by fluorescence in site hybridization (FISH))

Cell identification and sorting

( by flow cytometry )

Cell imaging

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XMUGXQ PFS01-ITF011.1 Phenomenon of Fluorescence♦ 16 century, Spanish scientist, N. Monardes, observed Lignum Nephritium’s fluorescence

♦ 17 century, English scientist, Boyle and Newton, both observed fluorescence

♦ 18 century, English (Germany) scientist, John Herschel, observed fluorescence phenomenon of quinine

On a case of superfical color presented by a homogeneous liquid internally colorless. By Sir John Frederick William Herschel, Phillosophical transactions of the Royal Society of London (1845) 135:143-145

♦ 1852, Irish scientist, G.G. Stokes, re-studied John Herschel’s experiment, Observed Stokes’ shift, named fluorescence

XQGuo

spanish 内科医生N.Monardes观察到菲律宾紫檀木切片的黄色水溶液呈现出一种极为鲜艳的天蓝色。

XQGuo

As a footnote,Herschel wrote "I write from recollection of an experiment made nearlly twenty years ago, and which I cannot repeat for want of a specimen of the wood."

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The phenomenon of fluorescence described by Herschel

……Equal weights of the sulphate of quinine and of crystallized tartaric acid （酒石酸） , rubbed up together with addition of a very little water, dissolved entirely and immediately. It is this solution, largely diluted, which exhibits the optical phenomenon in question. Though perfectly transparent and colorless when held between the eye and the light, or a white object, it yet exhibits in certain aspects, and under certain incidences of the light, an extremely vivid and beautiful celestial blue color, which, from the circumstance of its occurrence, would seem to originate in those strata which the light first penetrates in the liquid, and which, if not strictly superficial, at least exert their peculiar power of analyzing the incident rays and dispersing those which compose the tint in question, only through a very small depth within the medium. ……

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1.2 Excitation and deactivation of molecule

1、用原子轨道线性组合法产生出各个分子轨道；

2、把电子加到每个分子轨道中去，在每个分子轨道中最多加进两个电子（ Pauli原理），由此产生分子的电子组态；3、把电子对加到最低能量轨道中去（建造原理），从而产生最低能量的电子组态（基态电子组态）例：甲醛的分子轨道

0CO

2O

2CO

2O

2CH

2CH

2O

2C

2O2

)*()()()(

)()()2()1()1()OCH(

n

SSS

C

最高占有分子轨道最低空轨道2

O2

CO2 )()()OCH( nK 电子基态

1.2.1 1.2.1 分子基态的电子组态分子基态的电子组态

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电子跃迁与电子激发态2

O2

CO2 )()()OCH( nK 甲醛的电子基态

C OH

H

..

..

′ COOn

*CO

*n *

COOn

*CO

*,n *, 22n

22n

*,n *,n

*, *,S2

S1

S0

T2

T1

电子跃迁类型

最低激发态和基态的电子组态最低激发态和基态的电子态

COOn

*CO

22n

电子多重态

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电子的多重态

h +

单重态（自旋配

对）

电子跃迁

激发单重态（自旋配对）

电子跃迁和自旋翻转

h +

单重态（自旋配对）

三重态（自旋平行）

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1.2.2 Jablonski diagram

T1

hA

hA

Absorption

10-15 s

Vibrational relax, VR, 10-1210-14 s

Internal conversion, IC, 10-1110-13 s

Intersystem crossing, ISC, 10-210-

6 s

Fluorescence

Phosphorescence

IC ISC

S0

S1

S2

3 2 1 0

10-8 s

10-3100 s

hF

hP

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Keys for understanding the Jablonski diagram

Deactivation processes

VR, vibrational relax

IC, internal conversion

ISC, intersystem crossing F, fluorescence

P, phosphorescenc

Time scale for each process

10-1210-14 s

10-1110-13 s

10-210-6 s

10-8 s

10-3100 s

Thermal equilibrum

Absorption, typically from lowest-energy vibrational state of S0

Emission, from lowest-energy vibrational state of S

1

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1.3 分子发光类型

激发模式光致发光

化学发光（生物发光）

场致发光

摩擦发光

热致发光电子激发态的类型

荧光

磷光

1.3.1 发光类型

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1.3.2 荧光的类型及其发射过程

Fhv hvSSSS A 0110

/0

teFF

瞬时荧光与迟滞荧光（比较荧光寿命）

瞬时荧光

常规荧光

荧光寿命 10-8 s

满足单指数衰变规律

激基二聚体荧光

F

Fhv

hvSSSSS

hvSSSS A

01101

0110

2)(

0

itieFF /满足双指数衰变规律

荧光寿命 10-8 s

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Excimer

Pyrene, first species observed forming excimer, mono-mer, with em= 377 nm, excimer, with em= 480 nm

Dyke, D.A. V.; Pryor, B.A.; Smith, P.G.; Topp, M. R., Nanosecond time-resolved fluorescence spectroscopy in the physical chemistry laboratory: Formation of the pyrene excimer in solution, J.Chem. Edu. 75(5) 1998,615-620

Pyrene 芘

XQGuo

The fluorescence of the excited monomer in violet, with excitation maxmium at 377 nm, and the spectrum has considerable structure due to transitions to difference vibrational levels of the ground state...... However, the blue fluorescnece from the excimer, with excitation maxium at 480 nm, exhibits a broad structureless band, reflecting the fact that the double molecule dose not have a bound ground state.

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Characteristics of Excimer

Concentration depending Emission spectra changed with concentration though the concentration changes did not affect the shape of the Absorption spectrum.

C

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Characteristics of ExcimerViscosity depending The lower viscosity of solution allows the solute molecules to diffuse more rapidly through solution, promoting the excimer formation reaction.

hetradecane

10-3 M

10-3 M10-4 M

10-2 M

10-4 M

10-5 Mhexane

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Characteristics of Excimer Double-exponential time-decay profile

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Time related fluorescence spectra

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迟滞荧光

hvSSTSA ISC 0111

hvSS

SSTSB TISC

01

01111

现象：发射波长与常规发射波长一致，但寿命却与磷光相同。种类：

单重激发态与三重激发态处于热平衡状态，荧光寿命与磷光寿命相当。

荧光寿命是磷光寿命的一半。

hvRRRRR

hvRRReRC

2

自由基与电子的复合

自由基之间的复合

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瞬时荧光与迟滞荧光

• 从分析的觉度，更关注瞬时荧光。

• 分析常应用的是常规荧光。

• 二聚体荧光应用为探针，表征微环境粘度等。

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Stokes 荧光、反 Stokes 荧光、共振荧光从激发光与发射光的波长相对位移比较分类

Stokes 荧光

反 Stokes 荧光

共振荧光

ex < em

ex > em

ex = em

伴随附加热能给激发态分子

多光子技术

hvex hvem

A F

hvex hvem

A F

hvex hvem

A F

共振荧光

在气体状态下

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Multiphoton excitation

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Emission spectra of DPH with one (287 nm) and three-photon (860 nm) (excitation

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Scatter

Rayleigh scatter

Ramen scatter

s = ex

s = ex Related to excitation ex

Fluorescence independent of ex

hvex hvem

A F

How to differicate Rayleigh scatter, Raman scatter, and fluorescence?

How to differicate Rayleigh scatter, Raman scatter, and fluorescence?

F

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1.4 Characteristics of fluorescence emission

• 1.4.1 stokes’ shift• 1.4.2

Emission spectra , independent of the ex

• 1.4.3 Mirror image rule

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1.4 Characteristics of fluorescence emission

1.4.1 stokes’ shift

On the change fo refrangibility of light, By G.G. Stokes, M.A., F. R. S., Fellow of pembroke college, and Lucasian Professor of Mathemmatics in the University of Cambrige. Philosophical Transactions of the Royal Society of London (1852) 142: 463-462.

On the change fo refrangibility of light, By G.G. Stokes, M.A., F. R. S., Fellow of pembroke college, and Lucasian Professor of Mathemmatics in the University of Cambrige. Philosophical Transactions of the Royal Society of London (1852) 142: 463-462.

Description of the experiment

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Experiment

The solution of quinine, though it appears to be perfectl

y transparent and colorless, like water, when viewed by t

ransmitted light, exhibits nevertheless in certain aspects,

and under certain incidences of the light, a beautiful cel

estial blue color. It appears from the experiments of Sir J

ohn Herschel that the blue color comes only from a stra

um of fluid of small but finite thichness adjacent to the s

urface by which the light inters. After passing through th

is stratum, the incident light, though not sensibly enfeeb

led nor colored, has lost the power of producing the sam

e effect, and therefore may be considered as in some wa

y or other qualitatively different from the original light.

XQGuo

The quinine solution is colorless because it absorbes in the UV, which we cannot see. the blue color comes only from a region near the surface. this is because the quinine solution was relatively concentrated and absorbed all of the UV in the first several millimeters. Hence,stokes observed the inner filter effect. After passing through the solution, the light was "enfeebled" light could no longer capable of causing the blue glow. this occurred beacause the UV was removed and the "enfeebled" light could no longer excited quinine.

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Energy loss between excitation and emission

Causes

VR, vibrational relax

IC, internal conversion

Solvent effects

Excited-state reaction

Internal or intra-molecule reaction of excited-states, causing loss of energy

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XMUGXQ PFS01-ITF01Excited-state reactionLosing energy by protonating

ERHRR Hhv

)(

em ’em ex < em < ’em

激发态的碱性增强

5- 羟基吲哚

pH = 7.0 ex = 295 nm, em = 330 nm

酸性介质 ex = 295 nm, em = 550 nm

在酸性介质发生质子化

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Excited-state reactionLosing energy by forming excimer

F

Fhv

hvSSSSS

hvSSSS A

01101

0110

2)(

0 ex < (F0) < (F’)

Cmonomer

excimer

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O

O

OH

O

O

OH

Losing energy by excited-state intramolecular proton transfer( 激发态分子内质子转移） ESIPT

可以观察到很大的 stokes’ shift

H H

_ +

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C

N

N

Losing energy by twisted intramolecular charge transfer (TICT),

DMABN, dual fluorescence was observed, normal band (B band), 350 nm, anomalous band, (A band), 450 nm, in medium polar solvent. Grabowski: TICT

Reference

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Reference

Fluorescence probes based on TICT states, W. Rettig and Rene’ Lapouyade, in “Topics in fluorescence spectroscopy” Vol. 4, Probe design and chemical sensing, ed. By J. R. Lakowicz, Plenum Press, NY, 1994, pp.109-150.

Fluorescence probes based on TICT states, W. Rettig and Rene’ Lapouyade, in “Topics in fluorescence spectroscopy” Vol. 4, Probe design and chemical sensing, ed. By J. R. Lakowicz, Plenum Press, NY, 1994, pp.109-150.

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Solvent effect激发态的电子分布不同于基态，从而它们的永久偶极距和激化率也有所不同，因此两者的溶剂化程度不同。溶剂松弛导致能量进一步损失。

a

b

0-0

hvA

c

d

0-0

hvF

Relaxation

Relaxation

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1.4.2 Emission spectra

hvex hvem

A F exceptions exist, such as fluorophores which exist in two ionization states, each of which displays distinct absorption and emission spectra. also, some molecules are known to emit from the S2

independent of the excitation wavelength

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1.4.3 Mirror image ruleCauses:Franck-Condon principleSimilarity of the vibrational energy levels of excited-state and ground state

Sp

ectr

a o

f p

eren

e

F

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Franck-Condon principleTransition occur in about 10-15 s, a time too short for significant displacement of nuclei. All electronic transitions are vertical. The largest transition probability in absorption is also most probable in emission.

Similarity of the vibrational energy levels

The spacing of the vibrational energy levels of the excited states is similar to that of the ground states.

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Observation of mirror image rule

/)(

A rigorous test of the mirror image rule requires that the absorption and emission spectra be presented in appropriate units.

Absorption

Emission3/)( F

Correcting spectra

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Exception to mirror image ruleExcitation from other excited states

F

The emission is the mirror image of the S0 S1 absorption, not of the total absorption spectrum.

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Geometric arrangement of nuclei in excited state

Nuclear displacements can occur prior to emission because of the relative long lifetime of the S1 state, which allows time for motion following the instantaneous process of absorption.

XQGuo

It seems likely that the individual rings become more coplanar in the excited state. as result, the emission spectrum is more highly structured than the absorption spectrum.

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Complexing

Emission spectra Emission spectra

Charge-transfer complexCharge-transfer complex

anthraceneanthraceneexciplexexciplex

The mirror image of absorption The mirror image of absorption

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Excited-state reactionexcimer

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Acid-base reaction, change pKa in the excited state, may or may not change the spectral profile.

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Excited-state reactionTICT

N

OO

R

Documents

20040200 XMUGXQ PFS01-ITF01 Principles of Fluorescence Spectroscopy Chemistry Department XMU