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University of Cyprus
Fluorescence S ectrosco
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Principles of Fluorescence
Luminescence Emission of photons from electronically
excited states Relaxation from singlet excited state Relaxation from triplet excited state
ng e an r p e s a es Ground state two electrons per orbital; electronshave opposite spin and are paired
Singlet excited state Electron in higher energy orbital has the opposite spin
orientation relative to electron in the lower orbital The excited valence electron may spontaneously
reverse its spin (spin flip). This process is calledintersystem crossing. Electrons in both orbitals now
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ave same sp n or en a on
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Principles of Fluorescence
Types of emission Energy level diagram Fluorescence
return from excited singletstate to ground state;
a ons agram
oes not requ re c angein spin orientation (morecommon of relaxation)
return from a tripletexcited state to a groundstate electron re uireschange in spin orientation
Emissive rates offluorescence are severalorders of magnitudefaster than that ofphosphorescence
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Principles of Fluorescence
The ratio of molecules in S1
n
nupper
kTnlower =exp
k=1.38*10-23 JK-1 (Boltzmanns constant)E = separation in energy level
nlower
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Principles of Fluorescence
Excitation Light is absorbed; for dilute
sample, Beer-Lambert law applies Magnitude of reflects probability of S1
a sorp on Wavelength of dependence
corresponds to absorption spectrum
( )A Cl == -1 -1
Franck-Condon principle
C = concentration, l = pathlength
The absorption process takes placeon a time scale (10-15 s) much fasterthan that of molecular vibration
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Principles of Fluorescence
Non-radiative relaxation The electron is promoted to
hi her vibrational level in S1S1 state than the vibrationallevel it was in at the ground
state Vibrational deactivation
takes place through
intermolecular collisions me sca e o - s as erthan that of fluorescencerocess
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Principles of Fluorescence
Emission The molecule relaxes from the
lowest vibrational energy level ofthe excited state to a vibrational S1energy level of the ground state(10-9 s)
lower than that of the incidentphotons
m ss on pec rum For a given excitation wavelength, the
emission transition is distributed among
For a single excitation wavelength, canmeasure a fluorescence emission
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Principles of Fluorescence
Emission Stokes shift
Fluorescence light is red-shifted (longerwavelength than the excitation light) S1
Internal conversion can affect Stokes shift Solvent effects and excited state reactions
Stokes shift Invariance of emission wavelength with
Emission wavelength only depends onrelaxation back to lowest vibrational level
of S1 For a molecule, the same fluorescence
emission wavelength is observedirrespective of the excitation wavelength
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Principles of Fluorescence
Mirror image rulev=5
Vibrational levels in the excited
states and ground states are
1
v=0
An absorption spectrumreflects the vibrational levels of
v=5
the electronically excited state An emission spectrum reflects S
t e v rat ona eve s o t eelectronic ground state
v=0
spectrum is mirror image ofabsorption spectrum
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Principles of Fluorescence
Internal conversion vs. fluorescence
Electronic energy increases --> the
energy levels grow more closely Overlap between the high vibrational
energy levels of Sn-1 and low vibrationalenergy levels of Sn more likely
s over ap ma es rans on e ween
states highly probable Internal conversion: a transition
between states of the same multi licit Time scale of 10-12 s (faster than that of
fluorescence process)
Significantly large energy gape ween 1 an 0 S1 lifetime is longer radiative emission
can compete effectively with non-radiative emission
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Principles of Fluorescence
Internal conversion vs. fluorescence emission Mirror-image rule typically applies when only S0 S1 excitation
takes place
Deviations from the mirror-ima e rule are observed when S S2 or transitions to even higher excited states also take place
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Principles of fluorescence
Intersystem crossing n ersys em cross ng re ers o
non-radiative transition betweenstates of different multiplicity
It occurs via inversion of the spino t e exc te e ectron resu t ng ntwo unpaired electrons with thesame spin orientation, resulting ina triplet state
Transitions between states ofdifferent multiplicity are formallyforbidden
S in-orbit and vibronic cou linmechanisms decrease the purecharacter of the initial and finalstates, making intersystemcrossing probable
T1 S0 transition is alsoforbidden T1 lifetimesignificantly larger than S1 lifetime10-3-102 s
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Quantum yield and life time
Quantum yield of fluorescence, f, is defined as:
number of photons emitted
number of photons absorbedf =
Another definition for f is
= kr
f
where kr is the radiative rate constant and k is the sum of the rateconstants for all processes that depopulate the S1 state.
In the absence of com etin athwa s =1 Characteristics of quantum yield
Quantum yield of fluorescence depends on biological environment Exam le: Fura 2 excitation s ectrum and Indo-1 emission s ectrum
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and quantum yield change when bound to Ca +
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Quantum yield and life time
Radiative lifetime, r, is related to kr
r
r
k
=
e o serve uorescence e me, s e average methe molecule spends in the excited state, and it is
1
Characteristics of life-time=
kf
rov e an a ona mens on o n orma on m ss ng ntime-integrated steady-state spectral measurements
Sensitive to biochemical microenvironment, including local, Lifetimes unaffected by variations in excitation intensity,
concentration or sources of optical loss
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Quantum yield and life time
Fluorescence life-time methods Short pulse excitation followed by an interval during
which the resulting fluorescence is measured as afunction of time
Provide an additional dimension of information
m ss ng n me- n egra e s ea y-s a e spec rameasurements ,
local pH, oxygenation and binding
intensity, concentration or sources of optical loss
Com atible with clinical measurements in vivo
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Fluorescence Intensity
Absorbed intensit for a dilute solution Very small absorbance
From the Beer-Lambert law
( ) ( ) ( ), ( ) 2.303x m A m o x mF I Z I CL Z = =
where, Z instrumental factor (collection angle) Io incident light intensity
molar extinction coefficient quan um y e C concentration
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Fluorescence Intensity
Emission spectrume
Fixed Excitation
ce
Fixed Emission
o exc a on wave eng xe ,scan emission
Reports on the fluorescencespectral profile luo
rescen
Flu
oresce
reflects fluorescence quantum yield,k(lm)
Excitation spectrum
Emission (nm)Excitation (nm)
550
Hold emission wavelength fixed,scan excitation
Reports on absorption structurenm)
450470490510 FAD
,(lx)
Excitation-Emission MatrixExcitation
370
390410430
NADH
Composite Good representation of multi-
fluorophore solution 290310330350
Tryp.
17Emission(nm)
300 350 400 450 500 550 600 650 700250
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Quenching, Bleaching & Saturation
Excited molecules relax to ground states via
emission (vibration, collision, intersystem
Molecular oxygen quenches by increasing the
Polar solvents such as water generally quenchfluorescence by orienting around the exited statedipoles
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Quenching, Bleaching & Saturation
Photobleaching Defined as the irreversible destruction of an excited
fluorophore
window for excitation is very short (a few hundredmicroseconds)
Excitation Saturation The rate of emission is dependent upon the time the
state lifetime f)
Optical saturation occurs when the rate of excitationexcee s e rec proca o Molecules that remain in the excitation beam for extended
eriods have hi her robabilit of interstate crossin s and
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thus phosphorescence
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Fluorescence Resonance Energy
Non radiative energy transfer a quantum mechanical process of
Effective between 10-100 only
Emission and excitation spectrum must significantly overlap onor rans ers non-ra a ve y o e accep or FRET is very sensitive to the distance between donor an acceptor and
is therefore an extremely useful tool for studying molecular dynamics
Molecule 1 Molecule 2Molecule 1 Molecule 2
DONOR ACCEPTOR
Absorbance Absorbance
uorescence uorescence
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WavelengthWavelength
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Fluorescence Recovery after Photo-
Useful technique for studying,
especially transmembrane proteindiffusion
FRAP can be used to estimate therate of diffusion, and the fraction ofmolecules that are mobile/immobile
Can also be used to distinguish
diffusion Procedure
a e e mo ecu e w afluorophore
Bleach (destroy) the fluorophore is a
intensity laser Use a weaker beam to examine the
recovery of fluorescence as aNature Cell Biology 3, E145 - E147 (2001)
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unct on o t me
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Biological Fluorophores
Endogenousuorop ores
amino acids enzymes and co-enzymes vitamins lipids porphyrins
xogenousFluorophores
C anine d es Photosensitizers Molecular markers GFP,
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.
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Biological Fluorophores
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Fluorescence Instrumentation
Fluorescence is a highly
analyte concentration of 10-8 M)
mpor an o m n m zeinterference from:
Background fluorescence fromsolvents
Light leaks in the instrument Stray light scattered by turbidsolutions
Instruments do not yield ideal
spectra: on-un orm spec ra ou pu o gsource
Wavelength dependent efficiency ofdetector and optical elemens
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Fluorescence Instrumentation
Major components foruorescence ns rumen Illumination source
Broadband (Xe lamp)
Excitation Emission
Control Monochromatic (LED, laser)
Light delivery to sample Lenses/mirrors
LightSource
p ca ers
Wavelength separation(potentially for both excitation
Monochromator
ImagingSpectrograph
Filters Monochromator
S ectro ra h
Fiber
Optic Detector
PMT CCD camera
Probe
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Applications
Has disease Does not have
Tests positive (A)
True positive
(B)
False positive
(A+B)
Total # who test
Tests negative (C)
False negative
(D)
True negative
(C+D)
Total # who test
(A+C)
Total # who have
(B+D)
Total # who do not
Sensitivity=A/(A+C)= +
Positive predictive value=A/(A+B)= +
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Applications
- 0.5 Datacancerous lesions
00.1
0.2
0.3
0.4
.
ectocervix 350 450 550 650
Wavelength (nm)
1.2 Pre-Processin Ste 1
00.20.40.60.8
1
endocervix
350 450 550 650
Wavelength (nm)
0.4 Pre-Processing Step 2
-0.2
0
0.2
350 450 550 650Normal squamous
Low- rade
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- .
Wavelength (nm)
High-grade
Normal columnar
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Applications
-cancerous lesions
SILs vs. NON SILs HG SIL vs. Non HG SIL
Classification Sensitivity Specificity Sensitivity Specificity
Pap Smear Screening 62% 23 68%21 N/A N/A
Colposcopy in Expert Hands 94%6 48%23 79%23 76%13
Full Parameter
Composite Algorithm82%1.4 68%0.0 79%2 78%6
-Composite Algorithm
84%1.5 65%2 78%0.7 74%2
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Applications
Detection of lung carcinoma in situ using the LIFEmag ng sys em
Carcinoma in situ
White light bronchoscopy Autofluorescence ratio
29Courtesy of Xillix Technologies (www.xillix.com)
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Applications
Detection of lung carcinoma in situ using the LIFEmag ng sys em
Autofluorescence enhances ability to localize smallneo lastic lesions
Severe dysplasia/Worse Intraepithelial Neoplasia
WLB WLB+LIFE WLB WLB+LIFE
Sensitivity 0.25 0.67 0.09 0.56
Positive predictive value 0.39 0.33 0.14 0.23
Negative predictive value 0.83 0.89 0.84 0.89
False positive rate 0.10 0.34 0.10 0.34
Relative sensitivity 2.71 6.3
30S Lam et al. Chest 113: 696-702, 1998
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Applications
Fluorescence lifetimemeasurements
Autofluorescencelifetimes measured fromcolon tissue in vivo
Analysis via iterative re-convolution
Two component modelwith double exponentialecay
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M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998
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Applications
Fluorescence lifetimemeasurements
Autofluorescencelifetimes used todistinguish
non-adenomatousol s in vivo
32M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998