Biomedical Optics

By: Success Kamuhanda

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 and triplet states
• 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
have same spin orientation

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Principles of Fluorescence

• Types of emission • Energy level diagram

• Fluorescence (Jablonski diagram)
• return from excited singlet
state to ground state;
does not require change
in spin orientation (more
common of relaxation)
• 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 S1
nupper

nupper
= exp − ∆E
nlower kT
So
nlower
k=1.38*10-23 JK-1 (Boltzmann’s constant)
∆E = separation in energy level

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Principles of Fluorescence

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

A =  ()Cl
ε = molar absorption coefficient (M-1 cm-1) So
C = concentration, l = pathlength
• Franck-Condon principle
• The absorption process takes place
on a time scale (10-15 s) much faster
than that of molecular vibration

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Principles of Fluorescence

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

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Principles of Fluorescence

• Emission
• The molecule relaxes from the
lowest vibrational energy level of
the excited state to a vibrational S1
energy level of the ground state
(10-9 s)
• The energy of the emitted photon is
lower than that of the incident
photons
• Emission Spectrum So
• 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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Principles of Fluorescence

• Emission
• Stokes shift
• Fluorescence light is red-shifted (longer
wavelength than the excitation light) S1
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
• Invariance of emission wavelength with
excitation wavelength So
• 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

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Principles of Fluorescence
v’=5
• Mirror image rule
S1
• Vibrational levels in the excited v’=0
states and ground states are
similar
v=5
• An absorption spectrum
reflects the vibrational levels of
the electronically excited state
• An emission spectrum reflects S0
the vibrational levels of the v=0
electronic ground state
• Fluorescence emission
spectrum is mirror image of
absorption spectrum

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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 overlap makes transition between
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
between S1 and S0
• 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-image rule are observed when S0 
S2 or transitions to even higher excited states also take place

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Principles of fluorescence

• Intersystem crossing
• Intersystem crossing refers to
non-radiative transition between
states of different multiplicity
• It occurs via inversion of the spin
of the excited electron resulting 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 and vibronic coupling
mechanisms decrease the “pure”
character of the initial and final
states, making intersystem
crossing 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, Φf, is defined as:

number of photons emitted
f =
number of photons absorbed
• Another definition for f is
kr
f =
k
• 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 pathways Φf=1
• Characteristics of quantum yield
• Quantum yield of fluorescence depends on biological environment
• Example: Fura 2 excitation spectrum and Indo2-+1 emission spectrum
and quantum yield change when bound to Ca

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Quantum yield and life time

• Radiative lifetime, τr, is related to kr

1
r =
kr
• The observed fluorescence lifetime, is the average time
the molecule spends in the excited state, and it is
1
f =
k
• Characteristics of life-time
• Provide an additional dimension of information missing in
time-integrated steady-state spectral measurements
• Sensitive to biochemical microenvironment, including local
pH, oxygenation and binding
• Lifetimes unaffected by variations in excitation intensity,
concentration or sources of optical loss
• Compatible with clinical measurements in vivo

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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 a
function of time
• Provide an additional dimension of information
missing in time-integrated steady-state spectral
measurements
• Sensitive to biochemical microenvironment, including
local pH, oxygenation and binding
• Lifetimes unaffected by variations in excitation
intensity, concentration or sources of optical loss
• Compatible with clinical measurements in vivo

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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
exceeds the reciprocal of f
• Molecules that remain in the excitation beam for extended
periods have higher probability of interstate crossings and
thus phosphorescence
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Biological Fluorophores

• Endogenous
Fluorophores
• amino acids
• structural proteins
• enzymes and co-enzymes
• vitamins
• lipids
• porphyrins
• Exogenous
Fluorophores
• Cyanine dyes
• Photosensitizers
• Molecular markers – GFP,
etc.

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Fluorescence Instrumentation

• Fluorescence is a highly
sensitive method (can measure
analyte concentration of 10-8 M)

• Important to minimize
interference from:
• Background fluorescence from
solvents
• Light leaks in the instrument
• Stray light scattered by turbid
solutions

• Instruments do not yield ideal

spectra:
• Non-uniform spectral output of light
source
• Wavelength dependent efficiency of
detector and optical elemens

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Fluorescence Instrumentation

• Major components for

fluorescence instrument
• Illumination source Excitation Emission
• Broadband (Xe lamp) Control
• Monochromatic (LED, laser) CCD
Light
• Light delivery to sample Source
• Lenses/mirrors
• Optical fibers
• Wavelength separation Mono Imaging
(potentially for both excitation and chromator Spectrograph
emission)
• Filters
• Monochromator Fiber
• Spectrograph Optic
Probe
• Detector
• PMT
• CCD camera

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Applications

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

Sensitivity=A/(A+C) Positive predictive value=A/(A+B)

Specificity=D/(B+D) Negative predictive value=D/(C+D)

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Applications

• Detection of cervical pre- 0.5 Data

0.4

cancerous lesions 0.3

0.2
0.1
0
350 450 550 650
ectocervix Wavelength (nm)

1.2 Pre-Processing Step 1

1
0.8
0.6
0.4
0.2
0
350 450 550 650
Wavelength (nm)
endocervix
0.4 Pre-Processing Step 2
0.2
0
Normal squamous -0.2 350 450 550 650
Low-grade
-0.4
High-grade
Normal columnar Wavelength (nm)
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Applications

• Detection of cervical pre-

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 82%±1.4 68%±0.0 79%±2 78%±6

Composite Algorithm
Reduced-Parameter
84%±1.5 65%±2 78%±0.7 74%±2
Composite Algorithm

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Applications

• Detection of lung carcinoma in situ using the LIFE

imaging system
Carcinoma in situ

White light bronchoscopy Autofluorescence ratio

image

Courtesy of Xillix Technologies (www.xillix.com) 29

 Applications

• Detection of lung carcinoma in situ using the LIFE

imaging system
• Autofluorescence enhances ability to localize small
neoplastic 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

S Lam et al. Chest 113: 696-702, 1998 30

 Applications

• Fluorescence lifetime
measurements
• Autofluorescence
lifetimes measured from
colon tissue in vivo
• Analysis via iterative re-
convolution
• Two component model
with double exponential
decay

M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998 31

 Applications

• Fluorescence lifetime
measurements
• Autofluorescence
lifetimes used to
distinguish
adenomatous from
non-adenomatous
polyps in vivo

M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998 32

Factors Affecting Quantitative Accuracy

Non Linearity
• The proportional relationship between light absorption and
fluorescence emission is only valid for cases where the
absorption is small.
• As the concentration of fluorophore increases, deviations
occur and the plot of emission against concentration
becomes non-linear.
• With right-angle viewing, the principal distortion arises from
the absorption of the excited light before it can penetrate to
the heart of the cell, where the emission produced is
accepted by the detector optics
Temperature Effects

• Changes in temperature affect the viscosity of the medium and

hence the number of collisions of the molecules of the
fluorophore with solvent molecules.
• Fluorescence intensity is sensitive to such changes and the
fluorescence of many certain fluorophores shows a temperature
dependence. In such cases the use of thermos-tatted cell
holders is to be recommended.
• Normally it is sufficient to work at room temperature with the
provision that any sample procedure involving heating or
cooling must also allow sufficient time for the final solution to
reach ambient before measurement
PH Effects

• Relatively small changes in pH will sometimes radically affect the

intensity and spectral characteristics of fluorescence.
• Accurate pH control is essential and, when particular buffer
solutions are recommended in an assay procedure, they should
not be changed without investigation.
• Most phenols are fluorescent in neutral or acidic media, but the
presence of a base leads to the formation of non-fluorescent
phonate ions.
• 5 hydroxyl-indoles, for example, serotonin, show a shift in
fluorescence emission maximum from 330 nm at neutral pH to
550 nm in strong acid without any change in the absorption
spectrum
Inner Filter Effects

• Fluorescence intensity will be reduced by the presence of any

compound which is capable of absorbing a portion of either the
excitation or emission energy.
• At high concentrations this can be caused by absorption due to the
fluorophore itself.
• More commonly, particularly when working with tissue or urine
extracts, it is the presence of relatively large quantities of other
absorbing species that is troublesome.
• The purpose of extraction procedures is usually to eliminate such
species so that the final measurement is made upon a solution
essentially similar to the standard.
Quenching

• Decrease of fluorescence intensity by interaction of the

excited state of the fluorophore with its surroundings is
known as quenching and is fortunately relatively rare.
Quenching is not random.
• Each example is indicative of a specific chemical
interaction, and the common instances are well known.
• Quinine fluorescence is quenched by the presence of
halide ion despite the fact that the absorption spectrum
and extinction coefficient of quinine is identical in 0.5M
H2SO4 and 0.5M HCl