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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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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

3


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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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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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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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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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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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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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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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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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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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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

270


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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

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