fluorescence theory - latvijas universitātes...
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Fluorescence TheoryFluorescence Theory
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Fluorescence BasicsFluorescence Basics
! Fluorescence definition
! The fluorescence process
! Molar extinction coefficients
! Quantum yield
! Brightness
! Fluorescence lifetime
! FRET
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FluorescenceFluorescence
Spontaneous emission of radiation (luminescence)from an excited molecular entity with the formationof a molecular entity of the same spin multiplicity*
*From International Union of Pure and Applied Chemistry Glossary of Terms used inPhotochemistry (http://www.unibas.ch/epa/glossary/glossary/htm)
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Fluorescence DefinitionFluorescence Definition
! Fluorescence can be more simply defined as“the molecular absorption of light energy (photon) at onewavelength and its re-emission at another, usually longer,wavelength”
! Molecules which are able to absorb light are known aschromophores
! Molecules which are able to absorb and emit light are known asfluorochromes or fluorophores
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The Fluorescence ProcessThe Fluorescence Process
! The fluorescence process can be broken down into three phases
1. Excitation - absorption of light of an appropriate wavelength by fluorophore
2. Excited state - fluorophore undergoes vibrational and conformational changes
3. Emission - photon of light is emitted
! The fluorescence process is cyclical therefore a fluorophore can beexcited repeatedly
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ExcitationExcitation
Absorption of a photon and thus excitation to S1 or Sn respectively
S1
S0
Sn
Absorption
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Cy3 Excitation SpectrumCy3 Excitation Spectrum
Near UV Visible Near IR
400 450 500 550 600 650 700 7500
20
40
60
80
100
nm
Fluo
resc
ence
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Excited StateExcited State
S1
S0
Sn
Absorption
Radiationless energy loss to return to S1V1
Absorption of a photon and thus excitation to S1 or Sn respectively
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Fluorescence Emission Fluorescence Emission
Radiationless energy loss to return to S1
Absorption of a photon and thus excitation to S1 or Sn respectively
Reconversion to S0 from S1 withemission of radiation - fluorescence
S1
S0
Sn
Absorption
Emission
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Cy3 Emission SpectrumCy3 Emission Spectrum
400 450 500 550 600 650 7000
20
40
60
80
100
nm
Fluo
resc
ence
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Competing ProcessesCompeting Processes
ET Energy transfer to a nearby chromophore
ISC Intersystem crossing to triplet stateenergy dissipated via radiative (phosphorescence) or non-radiative pathways
S1
S0
IC ETT1
Phosphorescence
IC
ISC
Fluorescence
IC Radiationless internal conversion to the ground state
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Extinction Coefficient (εεεε)Extinction Coefficient (εεεε)
! The molar extinction coefficient is a measure of the lightabsorbing capacity of a dye - dyes with large molar extinctioncoefficients are efficient absorbers
! The molar extinction coefficient is required when determining theconcentration of a dilute solution of fluorophore using the Beer-Lambert law
Where A is absorbanceεεεε is molar extinction coefficientc is the concentration of the absorbing speciesl is the absorption path length
A = εεεεcl
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Quantum Yield (φφφφ)Quantum Yield (φφφφ)
! The fluorescence quantum yield is the ratio between the numberof fluorescence photons emitted and the number of photonsabsorbed:
absorbed photons ofnumber emitted photons ofnumber =φ
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BrightnessBrightness! Brightness is proportional to the product of the extinction
coefficient and quantum yield
Brightness }}}} εφεφεφεφ
! The brightness of a fluorophore labelled molecule isproportional to the extinction coefficient, quantum yield andnumber of dyes per molecule
Brightness }}}} nεφεφεφεφ
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Relative BrightnessRelative Brightness
Cy5
Cy3
Fluorescein
Non-sulphonated cyanine dyeTR
XRITC
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Fluorescence Lifetime (ττττ)Fluorescence Lifetime (ττττ)
! The fluorescence lifetime is the mean time spent in the excitedstate
! Natural or intrinsic fluorescence lifetime (τf)– Theoretical
! The excited state fluorescence lifetime (τex)– Measured value
! Excited state lifetimes can change with changes in fluorophoreenvironment
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Fluorescence Lifetime (ττττ)Fluorescence Lifetime (ττττ)
If(0)/e
If(0)
oo
τf
t
FLUORESCENCE DECAY CURVE
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FRETFRET
! Is a through space transfer of excitation energy from a donorfluorophore to an acceptor
! Can occur over distances of 10 - 100Å (1 - 10nm)
! The donor and acceptor must be spectrally related
! There is no emission of light by the donor
! The acceptor may or may not be fluorescent
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Fluorescence Resonance Energy TransferFluorescence Resonance Energy Transfer
S1
S0
Sn
Absorption
Emission
ICET
Donor
Emission
IC
Acceptor
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Energy Transfer Efficiency (E)Energy Transfer Efficiency (E)
R0 = Distance at which energy transfer efficiency is 50%r = Distance between donor-acceptor (Å)
+
= 660
60
rRRE
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R0 ValueR0 Value
( ) 61423
0 108.9 −×= nJR dφκ
R0 = Distance at which energy transfer efficiency is 50% J = Spectral overlap integral (extinction coefficient of acceptor buried within J)κ2 = Orientation factorφd = Donor quantum yield (in absence of acceptor)n = Solvent refractive index
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Cy3 and Cy5 SpectraCy3 and Cy5 Spectra
400 450 500 550 600 650 700 750 8000
20
40
60
80
100
Cy3 Cy5
ExcitationEmission
Wavelength (nm)
Flu
ores
cenc
e
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Dependence on Spectral Overlap (J)Dependence on Spectral Overlap (J)
0 20 40 60 80 100 1200
20
40
60
80
100
Dye (55.5)
Red-shifted (61.6)
Blue-shifted (49.1)
R0
R0
Distance (Å)
ET
eff
icie
ncy
(%)
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SummarySummary
! Define fluorescence
! The fluorescence process
! Molar extinction coefficients
! Quantum yield
! Brightness
! Fluorescence lifetime
! FRET
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Fluorescence ModulesFluorescence Modules
! Fluorescence Basics
! CyDye™ Chemistry
! CyDye Applications
! CyDyes and Fluorescence Polarisation
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Further InformationFurther Information
Can be found at Amersham Biosciences “Drug Screening Application Zone”
http://www.amershambiosciences.com
SPA (password prompt) →→→→ SPA
! Fluorescence theory: Fluorescence Overview
! FRET: Energy Transfer
! CyDye reagents: CyDye Fluor