principle of fluorescence
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Principle of fluorescence. Outline. Luminescence : fluorescence or phosphorescence? Jablonski diagram Characteristics of fluorescence emission Fluorescence lifetime and quantum yield Quantum mechanic behind Quenching Beer-Lambert law Biochemical fluorescence. phosphorescence. - PowerPoint PPT PresentationTRANSCRIPT
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Principle of fluorescence
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Outline• Luminescence : fluorescence or phosphorescence?• Jablonski diagram• Characteristics of fluorescence emission• Fluorescence lifetime and quantum yield• Quantum mechanic behind• Quenching• Beer-Lambert law• Biochemical fluorescence
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phosphorescence• Phosphorescence – electron go back to
ground state from triplet excited state (which is forbidden). Thus, it has lower rate about 103~100 s-1 (life time≈ms~s)
Pic. from : http://www.glassner.com/andrew/cg/research/fluphos/fluphos.htm
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Fluorescence
• Fluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetime≈ns).
Pic. From : http://en.wikipedia.org/wiki/Image:Fluorescent_minerals_hg.jpg
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Jablonski diagram
• Jablonski diagram can schematically tell us the fluorescence activity. It is proposed by Professor Alexander Jablonski in 1935 to describe absorption and emission of light.
Time scale (s)
Absorption 10-15
IC 10-12
ISC 10-8
F 10-8
P 10-3-100
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π-bond
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Mirror image
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Exception of mirror image
• Relaxation time is much smaller than emission, ΔE is much bigger than emission.
• Excimer – excited state dimer.• Influence of solvent – pH, O2…et
fluorescein
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Emission characteristic – Stoke’s shift
• Obviouly, form the Jablonski diagram of previous page, we know energy of emission light is less than energy of absorption light.
• This energy shift is called “Stoke’s shift”, usually shown in diagram by wavelength or wavenumber difference.
• Q1 and Q0 are energies of vibration taken by surround molecules.Q1≈Q0
hvF hvA (Q1 Q0) /NA
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Stoke’s shift
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Fluorescence lifetime
• First order rate equation!
• Unfortunately, there’s also contained nonradiative decay in nature.
• ko-1 is called natural lifetime, (ko+knr)-1 is real
lifetime.
d[ f ]
dt k0[ f ] [ f ] [ f ]0
k0te
d[ f ]
dt (k0 knr)[ f ] [ f ] [ f ]0
(k0 knr )t
e
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Quantum yield
• Definition is is the ration of the number of photons emitted to the number of photons absorbed. That is emission efficiency.
• Quantum yield can be calculated from standard quantum yield.
Qphotons emitted
photons absorbed
k0
k0 knr
QQRI
IR
ODROD
n2
nR2
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Quantum yield of some fluorephores
Quantum Yield [Q.Y.] Standards
Q.Y.[%] Conditions for Q.Y. Measurement
Excitation [nm]
Cy3 4 PBS 540
Cy5 27 PBS 620
Cresyl Violet 53 Methanol 580
Fluorescein 95 0.1 M NaOH, 22°C 496
POPOP 97 Cyclohexane 300
Quinine Sulfate 58 0.1 M H2SO4, 22°C 350
Rhodamine 101 100 Ethanol,25°C 450
Rhodamine 6G 94 Ethanol 488
Rhodamine B 31 Water 514
Tryptophan 13 Water, 20°C 280
L-Tyrosine 14 Water 275
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Quenching
• Enery of excited state could be taken by other substance, this process is called fluorescence quenching.
• Collisional (dynamics) quenching and static (complex- forming) quenching are most often process in quenching.
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Collisional quenching
• Oxygen, halogen, amines, and electron-deficient molecule often act as quenchers.
• In simplest quenching, stern-volmer equation holds
KD is stern-volmer quenching constant, kq is bimolecular constant, τ0 is unquenched lifetime.
F0
F1KD[Q] 1 kq 0[Q]
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Static quenching
• Energy is taken by forming complex.
• Combine with collisional quenching
Ks [FQ]
[F][Q]
[F]0 [F]
[F][Q]
F0
F1Ks[Q]
F0
F(1KD[Q])(1Ks[Q])
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Modify Stern-Volmer plots
• Some of fluorphores are accessibile and some aren’t for quenchers.
F0 Fa Fb F Fa
1Ka[Q] Fb
F0
F F0
1
faKa[Q]
1
fa
, where fa FaF0
F0 F FaKa[Q]
1Ka[Q]
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Time scale of molecular processes in solution
• Is quenching rapidly happened? • Ex. quenching by O2, which has diffusion
coefficient 2.5 x 10-5 cm2/s. The average distance of an O2 can diffuse in 10ns is given by Eistein equation
• About 7 nm. Concentration of quenching would process is
• In 25 oC water, oxygen dissolve is about 10-4 M
x 2 2D 5*10 13cm
C Liter
(7nm)3 3.3*1021 0.5 *10 2M
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Optical density
• In optics, density is the transmittance of an optical element for a given length and a given wavelength.
• In fluorescence, opticaldensity indicates us the absorption of fluorescentsolution.
Optical density log10
I0I
d
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Beer-Lambert law
• Absorption of light go through a substance is proportional to the effective cross section(σ), concentration of molecules(n) and intensity(I).
• Rewrite the Beer-Lambert law
where c is concentration (M) and ε is the extinction coefficient (M-1cm-1)
dI
dx In ln
I0I
nd
lnI0I
nd log10
I0I
cd optical density
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Extinction coefficient
• Extinction coefficient is calibrated by a fluorescent solution with width 1 cm and concentration 1 mole per liter.
fluorphores Extinction coefficient (M-1cm-
1)
Rhodamine 6G 105,000
Rhodamine B 123,000
Cell tracker (BLue) 16,000
SYTOX 38,000
IF Iincantilog( cd) * (Quantum Yield)
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Inner filter effect (IFE)
• Solution with optical density absorbs not only excitation light but also emission light.
• Excitation IEF• Emission IEF – absorbs by solute or fluorphores• Correction of IFE could be wrote down in the
following formula
• Usually, solution with OD<0.05 avoids IFE.
IF Iabsorbanti logODex ODem
2
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Biochemical fluorophores
• Intrinsic fluorphores• Extrinsic fluorphores• DNA probes• Chemical sensing probes• Fluorscent protein
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Intrinsic fluorphores
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Intrinsic fluorphores
• Vitamine A – Retinol, in liver stellate cell and retina.
Retinol
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Extrinsic fluorphores
• Eg. FITC, rhodamine – conjugate with protein, dextran, antibody…etc. for labeling specific target.
wavelength
fluor
esce
nce
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Extrinsic fluorphores
• Different Stoke’s shift of rhodamine derivatives.
wavelength1.Fluorescein 2.Rhodamine 6G
3.Tetramethylrhodamine 4.Lissamine rhodamine B 5.Texas Red
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DNA probes
• Hoechst33342 (binding to minor groove of DNA)
Red: rhodamine dextran blue: hoechst33342
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Fluorescent protein
• GFP – Green fluorescence protein• Extracted from jellyfishAequorea victoria.• Vector contained DNA of GFP is used in celltransfection.
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GFP
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As a reporter
• GFP vector• Put in liposome• Place into cells by injection or fusion
• Use as NFkB reporter
EGFP vector