University of Cyprus Biomedical Imaging and Applied Optics

Fluorescence Spectroscopy

Principles of Fluorescence
Luminescence Emission of photons from electronically excited states Two types of luminescence:
Relaxation from singlet excited state Relaxation from triplet excited state

Singlet Si l t and dt triplet i l t states t t

Ground state two electrons per orbital; electrons have 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

Triplet excited state

The excited valence electron may spontaneously reverse its spin (spin flip). This process is called intersystem crossing. Electrons in both orbitals now h have same spin i orientation i t ti
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Principles of Fluorescence
Types of emission
Fluorescence
return from excited singlet state to ground state; d does not t require i change h in spin orientation (more common of relaxation)

Energy level diagram (J bl (Jablonski ki diagram) di )

Phosphoresence
return from a triplet excited state to a ground state; electron requires change in spin orientation

Emissive rates of fluorescence are several orders of magnitude faster than that of phosphorescence
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Principles of Fluorescence
Population of energy levels The ratio of molecules in upper and lower states

nupper

S1

nupper nlower

= exp E
JK-1

kT

)
nlower So

k=1.38*10-23

(Boltzmanns constant) E = separation in energy level

Principles of Fluorescence
Excitation Light is absorbed; for dilute sample, Beer-Lambert law applies
Magnitude of reflects probability of absorption b ti Wavelength of dependence corresponds to absorption spectrum
S1

A = ( )Cl
= molar absorption coefficient (M-1 cm-1) C = concentration, l = pathlength So

Franck-Condon principle
The absorption process takes place on a time scale (10-15 s) much faster than that of molecular vibration

Principles of Fluorescence
Non-radiative relaxation The electron is promoted to higher g vibrational level in S1 state than the vibrational level it was in at the ground state Vibrational deactivation takes place through intermolecular collisions
12 s (faster A time ti scale l of10 f10-12 (f t than that of fluorescence process)

S1

So

Principles of Fluorescence
Emission The molecule relaxes from the lowest vibrational energy level of the excited state to a vibrational energy level of the ground state (10-9 s) The energy of the emitted photon is lower than that of the incident photons Emission E i i S Spectrum t
For a given excitation wavelength, the emission transition is distributed among different vibrational energy levels For a single excitation wavelength, can measure a fluorescence emission spectrum
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S1

So

Principles of Fluorescence
Emission Stokes shift
Fluorescence light is red-shifted (longer wavelength than the excitation light) relative to the absorbed light Internal conversion can affect Stokes shift Solvent effects and excited state reactions can also affect the magnitude of the Stokes shift
S1

Invariance of emission wavelength with excitation wavelength

Emission wavelength only depends on relaxation back to lowest vibrational level of S1 For a molecule, the same fluorescence emission wavelength is observed irrespective of the excitation wavelength

So

Principles of Fluorescence
Mirror image rule Vibrational levels in the excited states and ground states are similar An absorption spectrum reflects the vibrational levels of the electronically excited state An emission spectrum reflects the h vibrational ib i l levels l l of f the h electronic ground state Fluorescence emission spectrum is mirror image of absorption spectrum
v=5 S1 v=0 v=5

v=0

S0

Principles of Fluorescence
Internal conversion vs. fluorescence emission Electronic energy increases --> the energy levels grow more closely spaced
Overlap between the high vibrational energy levels of Sn-1 and low vibrational energy levels of Sn more likely This Thi overlap l makes k transition t iti b between t states highly probable

Internal conversion: a transition between states of the same multiplicity

Time scale of 10-12 s (faster than that of fluorescence process)

Significantly large energy gap b t between S1 and d S0

S1 lifetime is longer radiative emission can compete effectively with nonradiative 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 g rule are observed when S0 Deviations from the mirror-image S2 or transitions to even higher excited states also take place

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Principles of fluorescence
Intersystem crossing
I Intersystem t t crossing i refers f to t non-radiative transition between states of different multiplicity It occurs via inversion of the spin of f the th excited it d electron l t resulting lti i in two unpaired electrons with the same spin orientation, resulting in a triplet state Transitions between states of different multiplicity are formally forbidden Spin-orbit p and vibronic coupling p g mechanisms decrease the pure character of the initial and final states, making intersystem crossing gp probable T1 S0 transition is also forbidden T1 lifetime significantly larger than S1 lifetime (10-3-102 s) ( )

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Quantum yield and life time

Quantum yield of fluorescence, f, is defined as:
f = number of photons emitted number of photons absorbed

Another definition for f is

f =

kr

where kr is the radiative rate constant and k is the sum of the rate constants for all processes that depopulate the S1 state.

In the absence of competing p g pathways p y f=1 Characteristics of quantum yield

Quantum yield of fluorescence depends on biological environment a p e Fura ua2e excitation c a o spec spectrum u a and d Indo-1 do emission e ss o spec spectrum u Example: 2 2+ and d quantum yield i ld change h when h b bound d to C Ca
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Quantum yield and life time

Radiative lifetime, r, is related to kr

The Th observed b d fluorescence fl lifetime, lif ti is i the th average time ti the molecule spends in the excited state, and it is 1 f = k Characteristics of life-time Provide P id an additional dditi l di dimension i of fi information f ti missing i i i in time-integrated steady-state spectral measurements Sensitive to biochemical microenvironment, including local pH oxygenation and binding pH, Lifetimes unaffected by variations in excitation intensity, concentration or sources of optical loss Compatible with clinical measurements in vivo
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1 r = kr

Quantum yield and life time

Fluorescence life-time methods Short pulse excitation followed by an interval during which the resulting fluorescence is measured as a function of time Provide an additional dimension of information missing i i i in ti time-integrated i t t d steady-state t d t t spectral t l measurements Sensitive to biochemical microenvironment microenvironment, including local pH, oxygenation and binding Lifetimes unaffected by variations in excitation intensity, concentration or sources of optical loss Compatible p with clinical measurements in vivo
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Fluorescence Intensity
Absorbed intensity y for a dilute solution Very small absorbance From the Beer-Lambert law

F ( x , m ) = I A (m ) Z = 2.303I o ( x ) CL ( m ) Z
where,
Z instrumental factor ( (collection angle) g ) Io incident light intensity molar extinction coefficient quantum t yield i ld C concentration L path length
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Fluorescence Intensity
Fluorescen nce

H Hold ld excitation it ti wavelength l th fi fixed, d scan emission Reports on the fluorescence spectral profile reflects fluorescence quantum yield, k(lm)

Excitation (nm)

Fluorescenc F ce Emission (nm)

Emission spectrum

Fixed Emission

Fixed Excitation

Excitation spectrum
Excitation( (nm)

Hold emission wavelength fixed, scan excitation Reports on absorption structure reflects molar extinction coefficient, coefficient (lx)

Excitation-Emission Matrix (EEM)

Composite Good representation of multifluorophore solution

550 530 510 490 FAD 470 450 50 430 NADH 410 390 370 350 330 Tryp. 310 290 270 250 300 350 400 450 500 550 600 650 700 Emission(nm)
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Quenching, Bleaching & Saturation

Quenching Excited molecules relax to ground states via nonradiative pathways avoiding fluorescence emission (vibration, collision, intersystem crossing) Molecular oxygen quenches by increasing the probability of intersystem crossing Polar solvents such as water generally quench fluorescence by orienting around the exited state dipoles
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Quenching, Bleaching & Saturation

Photobleaching Defined as the irreversible destruction of an excited fluorophore Photobleaching is not a big problem as long as the time window for excitation is very short (a few hundred microseconds) Excitation Saturation The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime f) Optical saturation occurs when the rate of excitation e ceeds the reciprocal of f exceeds Molecules that remain in the excitation beam for extended periods have higher p g p probability y of interstate crossings g and thus phosphorescence
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Fluorescence Resonance Energy Transfer (FRET)

Non radiative energy transfer a quantum mechanical process of resonance between transition dipoles Effective between 10-100 only Emission and excitation spectrum must significantly overlap Donor D t transfers f non-radiatively di ti l to t the th acceptor t FRET is very sensitive to the distance between donor an acceptor and is therefore an extremely useful tool for studying molecular dynamics Molecule 1
Fl Fluorescence
DONOR ACCEPTOR

Molecule 2
Fl Fluorescence Absorbance

Absorbance

Wavelength
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Fluorescence Recovery after Photobleaching (FRAP)

Useful technique for studying transport properties within a cell cell, especially transmembrane protein diffusion FRAP can be used to estimate the rate of diffusion, and the fraction of molecules that are mobile/immobile Can also be used to distinguish between active transport and diffusion Procedure Label L b l th the molecule l l with ith a fluorophore Bleach (destroy) the fluorophore is a well defined area with a high intensity laser Use a weaker beam to examine the recovery of fluorescence as a f function ti of f time ti

Nature Cell Biology 3, E145 - E147 (2001)

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Biological Fluorophores
Endogenous Fl Fluorophores h
amino acids structural proteins enzymes and co-enzymes vitamins lipids porphyrins

Exogenous E Fluorophores
Cyanine y dyes y Photosensitizers Molecular markers GFP, etc etc.
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Biological Fluorophores

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Fluorescence Instrumentation
Fluorescence is a highly sensitive method (can measure analyte concentration of 10-8 M) I Important t t to t minimize i i i interference from:
Background fluorescence from solvents Light leaks in the instrument Stray light scattered by turbid solutions

Instruments do not yield ideal spectra:

N Non-uniform if spectral t l output t t of f light li ht source Wavelength dependent efficiency of detector and optical elemens

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Fluorescence Instrumentation
Major components for fl fluorescence instrument i t t
Illumination source
Broadband (Xe lamp) Monochromatic (LED, laser)
Excitation Light Source Mono chromator Control Emission
CCD

Light delivery to sample

Lenses/mirrors Optical O ti l fibers fib

Wavelength separation (potentially for both excitation and emission)

Filters Monochromator Spectrograph p g p

Imaging Spectrograph

Detector
PMT CCD camera

Fiber Optic Probe

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Applications
Test definitions
Has disease Tests positive (A) True positive (C) False negative (A+C) Total # who have disease Does not have disease (B) False positive (D) True negative (B+D) Total # who do not have disease (A+B) Total # who test positive (C+D) Total # who test negative

Tests negative

Sensitivity=A/(A+C) Specificity=D/(B+D) Specificity D/(B+D)

Positive predictive value=A/(A+B) Negative predictive value=D/(C+D) value D/(C+D)

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Applications
Detection of cervical prepre cancerous lesions
ectocervix
0.5 0.4 0.3 0.2 0.1 0 350 450 550 Wavelength (nm)

Data

650

1.2 1 0.8 0.6 0.4 0.2 0 350

Pre-Processing Pre Processing Step 1

450 Wavelength (nm)

550

650

endocervix
0.4 0.2 0 -0.2 350 -0.4 04

Pre-Processing Step 2

Normal squamous Low-grade g High-grade Normal columnar

450

550

650

Wavelength (nm)
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Applications
Detection of cervical prepre cancerous lesions
Classification Pap Smear Screening Colposcopy in Expert Hands Full Parameter Composite Algorithm Reduced Parameter Reduced-Parameter Composite Algorithm SILs vs. NON SILs HG SIL vs. Non HG SIL Sensitivity Specificity Sensitivity Specificity 62% 23 94%6 82%1.4 84%1.5 68%21 48%23 68%0.0 65%2 N/A 79%23 79%2 78%0.7 N/A 76%13 78%6 74%2

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Applications
Detection of lung carcinoma in situ using the LIFE i imaging i system t

Carcinoma in situ

White light bronchoscopy

Autofluorescence ratio image

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Courtesy of Xillix Technologies (www.xillix.com)

Applications
Detection of lung carcinoma in situ using the LIFE i imaging i system t Autofluorescence enhances ability to localize small p lesions neoplastic
Severe dysplasia/Worse WLB WLB+LIFE Sensitivity Positive predictive value Negative predictive value False positive rate Relative sensitivity y 0.25 0.39 0.83 0.10 2.71 0.67 0.33 0.89 0.34 Intraepithelial Neoplasia WLB WLB+LIFE 0.09 0.14 0.84 0.10 6.3 0.56 0.23 0.89 0.34

S Lam et al. Chest 113: 696-702, 1998

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Applications
Fluorescence lifetime measurements Autofluorescence lifetimes measured from colon tissue in vivo Analysis via iterative reconvolution
Two component model with double exponential decay
M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998
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Applications
Fluorescence lifetime measurements Autofluorescence lifetimes used to distinguish adenomatous from non-adenomatous polyps p yp in vivo

M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998

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