principles of fluorescence spectroscopy
DESCRIPTION
Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Five. Quenching of Fluorescence. Quenching of fluorescence. 5.1 Introduction 5.2 Stern-Volmer equation 5.3 Modified Stern-Volmer equation 5.4 factors influencing quenching 5.5 quenching mechanisms - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/1.jpg)
20040300
XMUGXQ PFS0501
Principles of Fluorescence Spectroscopy
Chemistry Department
XMU
![Page 2: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/2.jpg)
20040300
XMUGXQ PFS0501
Chapter Five
Quenching of
Fluorescence
![Page 3: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/3.jpg)
20040300
XMUGXQ PFS0501
Quenching of fluorescence
5.1 Introduction
5.2 Stern-Volmer equation
5.3 Modified Stern-Volmer equation
5.4 factors influencing quenching
5.5 quenching mechanisms
5.6 Application
![Page 4: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/4.jpg)
20040300
XMUGXQ PFS0501
5.1 introduction Fluorescence Quenching
Any processes decreasing the fluorescence intensity
Excited-state reactions
Molecular rearrangements
Ground-state complex formation
Collision
Quencher
Any species causing the decrease in the fluorescence
General quencher
Specific quencher
![Page 5: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/5.jpg)
20040300
XMUGXQ PFS0501
Dynamic quenching and static quenching Dynamic quenching
Collision quenching
** QMMhvM QA
relaxation (10-12 s)
S0
S1
S1
hvA hvF knr
Q
Q
kq[Q]
![Page 6: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/6.jpg)
20040300
XMUGXQ PFS0501
Dynamic quenching
nrkΓ
1][
1
QkkΓ qnr
In general, without permanent change in the fluorophore
Diffusion control
No changes in the absorption spectrum
Decreasing the lifetime
Change into
Effected by viscosity of solvent
100 F
F
Intensify with temperature increasing
![Page 7: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/7.jpg)
20040300
XMUGXQ PFS0501
Static quenching
*21 MQMQQM AA hvorhv MQ* → MQ +
MQ* → MQ + hvF2
1*
1 FA hvMMhvM
relaxation (10-12 s)
S0
S1
S1
hvA1 hvF1 knr
M
relaxation (10-12 s)
S0
S1
S1
hvA2 hvF2 knr
MQ
![Page 8: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/8.jpg)
20040300
XMUGXQ PFS0501
Static quenching
Changing the absorption spectrum
How about excitation spectrum? Change? Or not change?How about excitation spectrum? Change? Or not change?
Depend on MQ emitting or not
nrkΓ
1No change in the lifetime
How about the effect of temperature?How about the effect of temperature?
Depend on the thermodynamic properties of M and MQ
Forming a new species
![Page 9: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/9.jpg)
20040300
XMUGXQ PFS0501
Quenchers
oxygen
halogens
Causing ISC
Aromatic and aliphatic amines
Forming excited charge-transfer complexes
Carboxyl groups
Nitroxides
Nitromethane and nitro compounds
Heavy atoms
more……
![Page 10: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/10.jpg)
20040300
XMUGXQ PFS0501
5.2 Stern-Volmer eqution
][10 QKF
FSV
F0 and F
[Q] Concentration of quencher
Fluorescence intensities in the absence and presence of quencher, respectively
KSV Stern-Volmer quenching constant, given by kq
0
KD dynamic quenching
KS static quenching
![Page 11: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/11.jpg)
20040300
XMUGXQ PFS0501
Dynamic quenching01
dt
dS
]][[][][][ 1110 SQkSkSΓSk qnrA ][
][][ 0
1 QkkΓ
SkS
qnr
A
For steady-state measurement
In the presence of quencher
In the absence of quencher
][][][ 110 SkSΓSk nrA
nr
A
kΓ
SkS
][][ 0
01
relaxation (10-12 s)
S0
S1
S1
hvA hvF knr
Q
Q
kq[Q]
nr
qnr
kΓ
QkkΓ
S
S
][
][
][
1
01
][1
][1
0 Qk
QkΓ
k
q
nr
q
![Page 12: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/12.jpg)
20040300
XMUGXQ PFS0501
Dynamic quenching
][1][
][0
1
01 QkS
Sq
][1][
][0
0
1
01 QkF
F
S
Sq
nrkΓ
10
For quantitative measurement
Stern-Volmer equation
][
1
QkkΓ qnr Because
][1 00 Qkq
Stern-Volmer equation
![Page 13: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/13.jpg)
20040300
XMUGXQ PFS0501
Dynamic quenching
][1 00 Qk
F
Fq
kq Bimolecular quenching constant
Typically, 11010 mol-1 L s-1
0 Lifetime in the absence of quencher
kq = f(Q)k0
f(Q) Quenching efficiency
k0The diffusion-controlled bimolecular rate constant
))((1000
40 QFQF DDRR
Nk
R Molecular radius
D Diffusion coefficients N Avogadro’s number
(RF+RQ) collision radius
![Page 14: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/14.jpg)
20040300
XMUGXQ PFS0501
Example
Oxygen quenches The fluorescence of tryptophan
25°C, O2, Dq = 2.5 10-5 cm2/s;
tryptophan, DF = 0.66 10-5 cm2/sThe collision radius
R = ( RF + Rq ) = 5 Å
k0 =1.2 1010 mol-1 L s-1
Measured KD = 32.5 mol-1 L
Given = 2.7 ns,
Thus kq =1.2 1010 mol-1 L s-1
Thus
f(Q) = kq/k0 = 1
How to measure?How to measure?
![Page 15: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/15.jpg)
20040300
XMUGXQ PFS0501
The effect of lifetime on quenching
20 F
F
KD =kq0According to Stern-Volmer equation
Typically, kq = 11010 mol-1 L s-1
Typical fluorescence lifetime 0 = 10-8 s
Thus, KD = 102 mol-1 L
When LmolK
QD
/01.01
][ %50
Typical phosphorescence lifetime 0 = 10-3 s
Thus, KD = 107 mol-1 L
20 F
FWhen LmolK
QD
/101
][ 7%50
What do these calculations suggest? What do these calculations suggest?
Using oxygen as the quencher
![Page 16: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/16.jpg)
20040300
XMUGXQ PFS0501
Static quenching
MQ
M*
M + Q
hvF
(MQ)*
KS
]][[
)][(
QM
MQKS
)][(][][ 0 MQMM
]][[
][][ 0
QM
MMKS
][
][][][ 0
M
MMQKS
][1][
][ 0 QKM
MS
According to F = Kc
][1][
][ 0 QKF
FSThus
May or may not fluoresce
![Page 17: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/17.jpg)
20040300
XMUGXQ PFS0501
Stern-Volmer constant
KD
KS
= kq0 Related with lifetime, controlled by diffusion
Formation constant
Increasing with temperature increasing
T↑
1.0
[Q]
F0/F
T↑
1.0
[Q]
F0/F T↑
1.0
[Q]
F0/F
Exothermal reactionEndothermal reaction
![Page 18: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/18.jpg)
20040300
XMUGXQ PFS0501
Combined dynamic and static quenching
Violation of Stern-Volmer equation
1.0
[Q]
F0/FSuggest the combination of dynamic and static quenching
M*
MQM + Q
hvF
(MQ)*kq[Q]
dyanmic quenching
static quenching
KS
![Page 19: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/19.jpg)
20040300
XMUGXQ PFS0501
F*
FQ
Correction to Stern-Volmer equation
0][
][
F
Ff s ][1
][
][1 0 QKF
F
f Ss
)/()[Q]
(0*
*
nrqnr,F
FD kΓ
Γ
kkΓ
Γ
Φ
Φf
F
FQ
the fraction of fluorescence, due to static quenching
F0
hv
F*
Q*
the fraction of fluorescence, due to dynamic quenching
)[Q]
(qnr
nr
kkΓ
kΓ
![Page 20: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/20.jpg)
20040300
XMUGXQ PFS0501
Correction to Stern-Volmer equation
][1)[Q]
(1
0 QkkΓ
kkΓ
f qnr
qnr
D
DS ffF
F
0
F*
FQ
F
FQ
F0
hv
F*
Q*
fs
fS.fD])[1])([1(
110
QKQK
ffF
F
DS
DS
![Page 21: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/21.jpg)
20040300
XMUGXQ PFS0501
Correction to Stern-Volmer equation
])[1])([1(0 QKQKF
FDS
]/[)1(][)( 0 QF
FQKKKKK SDSDapp
Kapp apparent Stern-Volmer constant
][1
][])[(1 2
QK
QKKQKK
app
SDSD
![Page 22: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/22.jpg)
20040300
XMUGXQ PFS0501
Correction to Stern-Volmer equation
][)(]/[)1( 0 QKKKKQF
FSDSD
]/[)1( 0 QF
F
[Q]
KDKS
KD+KS
![Page 23: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/23.jpg)
20040300
XMUGXQ PFS0501
Modified Stern-Volmer equation in interpreting “sphere of action”
)1000/)]exp([])[1(/0 NQQKFF D
Where is the volume of the sphere.
The radius of the sphere is slightly larger than the sum of the radii of the fluorophore and the quencher.
There exists a high probability that quenching will occur before these molecules diffuse apart.
![Page 24: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/24.jpg)
20040300
XMUGXQ PFS0501
Example 1
Oxygen quenching of tryptophan
0/
x F0/F
![Page 25: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/25.jpg)
20040300
XMUGXQ PFS0501
Example 2
Acrylamide( 丙烯酰胺) quenching of N-acetyl-L-tryptophan-amide(N- 乙酰 -L- 色氨酸酰胺)
F0/F
F0/FQ
■ 0
/
![Page 26: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/26.jpg)
20040300
XMUGXQ PFS0501
Example 3
From JRL. P.245
Acrylamide quenching of dihydroequilenin (DHE ,二氢马萘雌甾酮 ) in buffer containing 10% sucrose (蔗糖) at 11°C
![Page 27: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/27.jpg)
20040300
XMUGXQ PFS0501
Example 4
10-methylacridinium chloride quenching of guanosine-5’-monophosphate (鸟嘌呤核苷 -5‘- 单磷酸)
![Page 28: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/28.jpg)
20040300
XMUGXQ PFS0501
Example 4
![Page 29: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/29.jpg)
20040300
XMUGXQ PFS0501
5.4 factors influencing quenching
Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤
Base
Suger-phosphoatebackboog
Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶
Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤
Base
Suger-phosphoatebackboog
Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶
Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤
Base
Suger-phosphoatebackboog
Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶
Steric effect
NCH2CH3 Br
NCH2CH3 Br
Example 1
![Page 30: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/30.jpg)
20040300
XMUGXQ PFS0501
The ethidium bromide-DNA complex
Oxygen quenching of ethdium bromide fluorescence
Why smaller than 11010?
Why smaller than 11010?
NCH2CH3 Br
NCH2CH3 Br
![Page 31: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/31.jpg)
20040300
XMUGXQ PFS0501
Example 2
![Page 32: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/32.jpg)
20040300
XMUGXQ PFS0501 Charge effect
F
I
O2
![Page 33: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/33.jpg)
20040300
XMUGXQ PFS0501
Example 1
Copolymer 1 1% tryptophan + 99% glutamic acid
Copolymer 2 3% tryptophan + 97% lysine
At neutral pH glutamic acid nagatively charged
Lysine positive charged
What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively?
What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively?
![Page 34: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/34.jpg)
20040300
XMUGXQ PFS0501
Example 1
![Page 35: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/35.jpg)
20040300
XMUGXQ PFS0501
Example 2
氯化十二烷基三甲铵
十二烷基硫酸钠
![Page 36: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/36.jpg)
20040300
XMUGXQ PFS0501
Micro-environment
Fluorescence quenching of trypsinogen 胰蛋白酶原荧光猝灭
![Page 37: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/37.jpg)
20040300
XMUGXQ PFS0501
Different in micro-environment
1.0
[Q]
F0/F
Downward-curving Stern-Volmer plot
Partial quenching
Fin
Fex I
F0 = Fin,0 + Fex,0
In the absence of quencher
![Page 38: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/38.jpg)
20040300
XMUGXQ PFS0501
Modified Stern-Volmer equation in interpreting the difference in micro-environment
][10, QKF
FD
ex
ex
0,0,
0, ][1 inD
exinex F
QK
FFFF
In the presence of quencher
Total fluorescence
0,0,
0,0,0 ][1 inD
exinex F
QK
FFFFFF
![Page 39: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/39.jpg)
20040300
XMUGXQ PFS0501
Deriving modified equation
][10,
0,0 QK
FFFFF
D
exex
][1
][ 0,0,0,
QK
FFQKF
D
exexDex
][1
][0, QK
QKF
D
Dex
![Page 40: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/40.jpg)
20040300
XMUGXQ PFS0501
Deriving modified equation
][1][
0,
0,0,0
QKQK
F
FF
F
F
D
Dex
inex
0,0,
0,
inex
exex FF
Ff
Let
Then
exDex fQKfF
F 1
][
10
Modified Stern-Volmer equation
![Page 41: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/41.jpg)
20040300
XMUGXQ PFS0501
Deriving modified equation
F
F
0
1/[Q]
1 /(KDfex)
1 / fex
exDex fQKfF
F 1
][
10
![Page 42: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/42.jpg)
20040300
XMUGXQ PFS0501
Example
Iodid
e qu
ench
ing of tryp
toph
an fl
uorescen
ce in lysozym
e(
溶菌酶)
Denatured protein
Native protein
Native protein
1/fex = 1.5, fex = 0.66
Denatured protein
1/fex = 1.0, fex = 1.0
![Page 43: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/43.jpg)
20040300
XMUGXQ PFS0501
example
![Page 44: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/44.jpg)
20040300
XMUGXQ PFS0501
Localization of membrane-bound fluorophores
![Page 45: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/45.jpg)
20040300
XMUGXQ PFS0501
Localization of membrane-bound fluorophores
![Page 46: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/46.jpg)
20040300
XMUGXQ PFS0501
5.5 quenching mechanisms
Nonradiative energy transfer, due to dipole-dipole interaction of donor and acceptor
5.5.1 Due to energy transfer
D* +A D+ A*
hvA hvF
D
D donor
A acceptor
Rate of energy transfer depends
Overlap of spectra
Relative orientation
Distance between A and D
![Page 47: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/47.jpg)
20040300
XMUGXQ PFS0501
Principle of energy
JNrn
kK
D
DT 642
2
128
)10(ln9000
Rate of energy transfer
D quantum yield of D
D lifetime of D
r distance between A and D
n refrcative index
N Avogadro’s number
k orientation factor
J overlap integral
![Page 48: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/48.jpg)
20040300
XMUGXQ PFS0501
Overlap integral
dFvd
v
vvFJ AD
AD
0
4
04
)()()()(
)(DF The corrected fluorescence intensity of D in -d, the total intensity normalized to unity
)( A The extinction coefficient of A at
The unit is (mol / L)-1 cm3
![Page 49: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/49.jpg)
20040300
XMUGXQ PFS0501
Orientation factor
k = 2/3
randomize by rotational diffusion prior to energy transfer
![Page 50: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/50.jpg)
20040300
XMUGXQ PFS0501
Förster distance
R0
relaxation (10-12 s)
S0
S1
S1
hvA hvF knrA(S0)
A(S1
)
TK
D1
The distance between A and D when
DnrT kΓK
1
R0 > r, energy transfer decay dominate
R0 < r, usual radiative and nonradiative decay dominate
![Page 51: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/51.jpg)
20040300
XMUGXQ PFS0501
Förster distance
254
260 108.8
n
JkR D
60 )(1
r
RK
DT
6
1
rKT Rate of energy transfer
Efficieney of energy transfer DnrDT
T
kΓK
KE
,
D
DA
FFE 1
1)/(
)/(6
0
60
rR
rRE
(in cm)
![Page 52: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/52.jpg)
20040300
XMUGXQ PFS0501
Example
聚脯氨酸
- 萘
D
丹磺酰
A
n =1-12, r = 12- 46Å
0% transfer
100% transfer
Excited D, measure F of A
no DF290= 2.3I0bcAA(A+ED)
A’= A+ED
D
AAE
'
![Page 53: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/53.jpg)
20040300
XMUGXQ PFS0501
Example
A
AD
A
DA
F
F ,
n =1, 100% transfer, the ratio of absorbance = the ratio of emission
![Page 54: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/54.jpg)
20040300
XMUGXQ PFS0501
example
1)/(
)/(
0
0
x
x
rR
rRE
01 lglg)1lg( RxrxE
x = 5.9±0.3
![Page 55: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/55.jpg)
20040300
XMUGXQ PFS0501
Sensitized fluorescence and phosphorescence
)()()()( 1001 SASDSASD
)()()()( 1001 TASDSASD
)()()()( 1001 SASDSATD
D(S1
)
D(S0
)
D(T1)A(T1)
A(S1)
A(S0)
)()()()( 1001 TASDSATD
Sensitized fluorescence
Sensitized phosphorescence
For D, quenched
For A, sensitized
![Page 56: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/56.jpg)
20040300
XMUGXQ PFS0501
ExampleSelf-association of ATPase (腺苷三磷酸酶) molecules in lipid vesicles
Mg2+-Ca2+-ATPase labeled individually with IAEDANS and IAF
Donor Acceptor
![Page 57: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/57.jpg)
20040300
XMUGXQ PFS0501
Intra-molecule energy transfer
![Page 58: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/58.jpg)
20040300
XMUGXQ PFS0501
Intra-molecule energy transfer
Trp
HSA
Tyr:Trp=18:1
R0 = 14Å
Comparable to the diameter of most proteins
![Page 59: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/59.jpg)
20040300
XMUGXQ PFS0501
5.5.2 duo to photochemical reaction
FF
hv
vhhv
RPRR A
**
荧光猝灭所涉及的光化学反应类型光化学反应荧光猝灭是指沿着激发态超平面发生的反应,导致激发态聚集数减少而引起的荧光猝灭。单分子光化学反应
绝热光化学反应 产生另一种形态的发光分子,其发射波长不同于原来的发光体
TICT
非绝热光化学反应 产生另一种具有稳定基态的物质,不伴随光子的发射
F
hv
hv
PPRR A **光分解
![Page 60: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/60.jpg)
20040300
XMUGXQ PFS0501
双分子光化学反应
F
RHhv
hv
RDHDD A 22*
激发态分子与其他分子发生反应,生成了稳定的或不稳定的新物质
光氧化还原 生成稳定的基态物质
激基二聚体 不具有稳定的基态
FF
Ahv
vhhv
AAAAA A
2*)(*
![Page 61: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/61.jpg)
20040300
XMUGXQ PFS0501
光化学反应的机制
R*
R
P*
P
S0
S1
hvF
hv’F
Excited state hypersurface
TICT
Dual fluorescence
R*
R
P*
P
S0
S1
hvFhv’F
Photoreaction
![Page 62: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/62.jpg)
20040300
XMUGXQ PFS0501
光化学反应的机制R*
R
P*
P
S0
S1
hvF
P’
Maurice R. Eftink, Fluorescence quenching: theory and application in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 53-120.
Herbert C. Cheung, Resonance energy transfer, in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 128-171.
Maurice R. Eftink, Fluorescence quenching: theory and application in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 53-120.
Herbert C. Cheung, Resonance energy transfer, in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 128-171.
![Page 63: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/63.jpg)
20040300
XMUGXQ PFS0501
5.6 Application
Molecular beacons
A D
Q F
Target DNA
ssDNA binding protein
Denaturing reagent
QF
F
Q
![Page 64: Principles of Fluorescence Spectroscopy](https://reader030.vdocument.in/reader030/viewer/2022033004/56813051550346895d96009c/html5/thumbnails/64.jpg)
20040300
XMUGXQ PFS0501
Molecular beacons
Hairpin probe show no fluorescence, do not need separation
Key type, almost no background fluorescence
Specific probe
Reference
Xiaohong Fang, Tianwei Heffery, John Perlette, Weihong Tan (University of Florida, USA) Kemin Wang (Hunan University, P.R. CHINA), Anal. Chem. 2000, Dec.1 747A-753A
Reference
Xiaohong Fang, Tianwei Heffery, John Perlette, Weihong Tan (University of Florida, USA) Kemin Wang (Hunan University, P.R. CHINA), Anal. Chem. 2000, Dec.1 747A-753A