Lecture 15

Fluorescence spectroscopy and

imaging:
Basic principles and sources of
contrast
Outline for Fluorescence
I. Princip-Prisip dasar Fluoresensi
II. Quantum Yield dan Lifetime
III. Spektroskopi Fluoresensi
IV. Fluorophores Biologik
V. Instrumentasi Fluoresensi
VI. Pengukuran Fluoresensi

I. Princip-Prinsip Dasar Fluoresensi
1. Luminesensi
Emisi fotons dari keadaan tereksitasi electronik

Dua tipes luminesensi:
Relakasasi dari keadaan eksitasi singlet excited
Relakaksasi dari keadaan eksitasi triplet



I. Princip Dasar Fluoresensi
2. Keadaan singlet dan triplet states
Keadaan dasar dua electron per orbital; electron
punya spin berlawanan dan berpasangan

Keadaan eksitasi singlet
Electron pada orbital energi lebih tinggi memiliki arah spin
berlawanan relative thd electron dalam orbital lebih rendah

Keadaan eksitasi triplet
Elektron valence tereksitasi secara spontan berbailk arah
spinnya (spin flip). Proes ini disebut intersystem crossing.
Electrons dlm kedua orbital sekarang memiliki arah spin sama

I. Princip Dasar Fluoresensi
3. Jenis emisi
Fluoresensi kembali dari keadaan eksitasi singlet ke
keadaan dasar; tidak memerlukan perubahan arah spin
(relaksasi yang lebih lazim)

Fosforesensi Kembali dari keadaan eksitasi triplet ke
keadaan dasar; electron perlu perubahan arah spin

Laji emisi fluoresensi beberapa tingkat lebih cepat
daripada fosforesensi (karena perubahan arah spin perlu
waktu)


I. Princip Dasar Fluoresensi



4. Diagram tingkat energy (Jablonski diagram)
I. Princip Dasar Fluoresensi
5a. Proses Fluoresensi : Population level energi
Pd suhu kamar (300 K), dan level energi electronic dan
vibrational tertentu, dpt dihitung ratio molekule dalam
tingkat lebih tinggi dan lebih rendah



( )
kT
E
n
n
lower
upper
A
= exp
k=1.38*10
-23
JK
-1
(Boltzmanns constant)
AE = perbedaan level energy
I. Prinsip Dasar Fluoresensi
5b. Proses Fluoresensi: Eksitasi
Pd suhu kamar, segala sesuatu berawal dari level energi
vibrational terendah pd keadaan dasar.

Jika sebuah molecule terpapar cahaya pada frekuensi
resonan, maka

Cahaya diserap; utk larutan encer , Hkm Beer-Lambert
berlaku
c koefisien absorpsi molar (extinction coeff)
(M
-1
cm
-1
); besarnya menggambarkan probabilitas serapan pada panjang
gelombang sesuai dengan pita absorpsinya.

Eksitasi: setelah penyerapan cahaya, suatu kromofor tereksitasi ke level
energi vibrational lbh tinggi, pada level S
1
atau S
2


Proses absorpti berlangsung pd skala waktu (10
-15
s) jauh lbh cepat d/p
vibrasi molekuler transisi vertical (Franck-Condon principle).
So
S1
cl A c =
E
n
e
r
g
y

nuclear configuration
I. Prinsip Dasar Fluoresensi
5c. Proses Fluoresensi: relaksasi Non-radiative
Dlm keadaan tereksitasi, elektron
dipromosikan ke orbital anti-bonding atom
dlm ikatan kurang kuat terikat bergeser ke
kanan kurva energi potential S
1
eletron
terpromosikan ke level energi vibrational
eksitasi S
1
lebih tinggi d/p level vibrational
dalam keadaan dasar.

Deaktivasi vibrational berlangsung lewat
tabrakan intermolekuler pd skala waktu
10
-12
s (lbh cepat d/p proses fluoresensi)
.


So
S1
I. Prinsip Dasar Fluoresensi
5d. Fluorescence process: Emission

Molekule relaksasi dari level vibrasional terendah
keadaan tereksitasi menuju level energi vibrasional
keadaan tereksitasi (10
-9
s)

Relaksasi ke keadaan dasar berlangsung lebih
cepat d/p waktu vibrasional molekuler
transisi vertical


Energy foton yg diemisikan lebih kecil
dibandingkan foton datang
So
S1
I. Prinsip Dasar Fluoresensi
6a. Pergeseran Stokes
Cahaya fluorescensi adalah red-shifted ( lbh panjang
d/p cahaya eksitasi) relatif thdp cahaya terabsorbsi.
("Stokes shift).

Konversi internal (see slide 13) dapat mempengaruhi
pergeseran Stokes (Stokes shift)

Solvent mempengaruhi reaksi keadaan tereksitasi dan juga
mempengaruhi besarnya Stokes shift


I. Prinsip Dasar Fluoresensi
6b. Invariansi panjang gelombang emission dgn
panjang gelombang eksitasi

emission hanya bergantung
pd waktu relaksasi ke level vibrasi
terendah pada level S
1

Utk molekul, emisi fluoresensi yg
sama muncul berapapun eksitasinya




So
S1
I. Prinsip Dasar Fluoresensi
6c. Aturan bayangan cermin
Level vibrational dlm keadaan tereksitasi
dan keadaan dasar adalah sama.

Spektra absorpsi merefleksikan level
vibrational keadaan eksitasi electronik

Spektra emisi merefleksikan level
vibrational electron keadaan dasar

Spektra emisi fluorescensi merupakan
bayangan cermin spektra absorpsi
S
0
S
1
v=0
v=1
v=2
v=3
v=4
v=5
v=0
v=1
v=2
v=3
v=4
v=5
I. Prinsip Dasar Fluoresensi
6d. Konversion Internal vs. Emisi fluoresensi

Jika energi electronic bertambah, level energi tumbuh more
closely spaced

Sangat boleh jadi akan overlap antara energi vibrasional
tinggi level S
n-1
and energi vibrational rendah levels of S
n


Overlap meyebabkan transisi antar states sangat mungkin

Konversi nternal mrpk transisi yg terjadi antara states dg
multiplisitas sama dan berlangsung pd skala waktu 10
-12

dt (lebih cepat d/p proses fluoresensi)

Perbedaan energi antara S
1
and S
0
cukup besar dibanding
antar states yg berdekatan lifetime S
1
lebih lama
emisi radiative dapat efektif sempurna dengan emisi non-
radiatif.

Mirror-image rule typically
applies when only S
0
S
1

excitation takes place
Deviations from the mirror-
image rule are observed when
S
0
S
2
or transitions to even
higher excited states also take
place
I. Prinsip Dasar Fluoresensi
6e. Intersystem crossing
Intersystem crossing adalah transisi non-radiative antar states (keadaan)yg
berbeda multiplisitasnya

Ini terjadi via inversi spin electron tereksitasi menghasilkan dua elektron tak
berpasangan dengan arah spin sama, memberikan keadaan dg spin = 1 dan
multiplisitas 3 (triplet state)

Transisi antar state berbeda multiplisitas pada dasarna adalah terlarang

Spin-orbit and vibronic coupling mechanisms decrease the pure character of
the initial and final states, memungkinkan intersystem crossing terjadi

Transisi T
1
S
0
juga forbidden lifetime T
1
sangat lebih besar d/p lifetime S
1

(10
-3
-10
2
s)
S
0
S
1
T
1
absorption
fluorescence
phosphorescence
Intersystem
crossing
I. Prinsip Dasar Fluoresensi
I. Prinsip Dasar Fluoresensi
Wavelength
Absorbance
DONOR
Absorbance
Fluorescence Fluorescence
ACCEPTOR
Molecule 1 Molecule 2
Fluorescence energy transfer (FRET)
Wavelength
Absorbance
DONOR
Absorbance
Fluorescence Fluorescence
ACCEPTOR
Molecule 1 Molecule 2
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 transfers non-radiatively to the acceptor

II. Quantum yield and lifetime
Quantum yield of fluorescence, u
f
, is defined as:





In practice, is measured by comparative measurements with reference
compound for which has been determined with high degree of accuracy.

Ideally, reference compound should have
the same absorbance as the compound of interest at given excitation wavelength
similar excitation-emission characteristics to compound of interest (otherwise,
instrument wavelength response should be taken into account)
Same solvent, because intensity of emitted light is dependent on refractive index
(otherwise, apply correction



Yields similar fluorescence intensity to ensure measurements are taken within the
range of linear instrument response
absorbed photons of number
emitted photons of number
= u
f
) (
) (
2
2
s n
u n
I
I
s
f
u
f
s
f
u
f
=
u
u
1a. Quantum yield of fluorescence
II. Quantum yield and life time
Another definition for u
f
is


where k
r
is the radiative rate constant and Ek is the sum of the
rate constants for all processes that depopulate the S
1
state.
In the absence of competing pathways u
f
=1
Radiative lifetime, t
r
, is related to k
r


The observed fluorescence lifetime, is the average time the
molecule spends in the excited state, and it is

= u
k
k
r
f
r
r
k
1
= t

=
k
f
1
t
1b. Fluorescence lifetime
II. Quantum Yield and Lifetime
2a. Characteristics of quantum yield
Quantum yield (hasil Kuantum) fluoresensi bergantung
pd lingkungan biologis
Contoh: spektrum eksitasi Fura 2 dan spektrum emisi
Indo-1 dan hasil quantum berubah ketika terikat pada
Ca
2+


Fura-2 changes in response to
varying [Ca2+]
Indo-1 changes in response to
varying [Ca2+]
II. Quantum yield and lifetime
2b. Characteristics of life-time
Provide an additional dimension of information
missing in time-integrated steady-state spectral
measurements
Sensitive thd mikrolingkungan biochemis, termasuk
pH lokal, oksigenasi dan ikatan
Lifetimes tak terpengaruh oleh variasi in intensitas
eksitasi, konsentrasi atau sumber hilangnya
keoptikan
Kompatible dg pengukuran klinik in vivo
Courtesy of M.-A. Mycek, U Michigan
II. Quantum Yield and Lifetime
3a. Fluorescence emission distribution
Utk eksitasi tertentu,
transisi emisi terdistribusi
di antara level energi
vibrational yg berbeda

Utk eksitasi tunggal
dpt mengukur suatu spectrum
emisi fluoresensi


I
n
t
e
n
s
i
t
y

Emission Wavelength (nm)
Exc
Emm
II. Quantum Yield and Lifetime
3b. Heisenbergs uncertainty principle
Values of particular pairs of observables cannot be
determined simultaneously with high precision in
quantum mechanics

Example of pairs of observables that are restricted in
this way are:

Momentum and position
Energy and time


II. Quantum Yield and Lifetime
3c. Heisenbergs uncertainty principle

Momentum and position :




Energy and time:


2
h
x p
x
> A A
2
h
t E > A A
II. Quantum Yield and Lifetime
3d. Effect on fluorescence emission
Suppose an excited molecule emits fluorescence in
relaxing back to the ground state

If the excited state lifetime, t is long, then emission will
be monochromatic (single line)

If the excited state lifetime, t is short, then emission
will have a wider range of frequencies (multiple lines)


I
n
t
e
n
s
i
t
y

Emission Wavelength (nm)
Exc
Emm
I
n
t
e
n
s
i
t
y

Emission Wavelength (nm)
Exc Emm
Large At small AE
Small At large AE
III. Fluorescence Intensity
1. Fluorescence intensity expression
2. Fluorescence spectra


III. Fluorescence Intensities
1a. Fluorescence intensity
The fluorescence intensity (F) at a particular excitation
(
x
) and emission wavelength (
m
) will depend on the
absorption and the quantum yield:



where,
I
A
light absorbed to promote electronic transition
| quantum yield


( ) ( ) ( )
m x A m x
I F | = ,
III. Fluorescence Intensities
1b. From the Beer-Lambert law, the absorbed intensity
for a dilute solution (very small absorbance)




where,
I
o
Initial intensity
c molar extinction coefficient
C concentration
L path length

1 << CL for
CL I 303 . 2 I
x
x o x A
c
c =
III. Fluorescence Intensities
1c. Fluorescence intensity expression
The fluorescence intensity (F) at a particular excitation
(
x
) and emission wavelength (
m
) for a dilute solution
containing a fluorophore is:



where,
I
o
incident light intensity | quantum yield
C concentration c molar extinction
L path length coefficient
( ) ( ) ( )
m x o m x
CL I F | c 303 . 2 , =
III. Fluorescence Intensities
1d. Measured fluorescence intensity
If we include instrument collection angle:



where,
Z instrumental factor
I
o
incident light intensity
c molar extinction coefficient
C = concentration
L path length
( ) ( ) ( )Z CL I F
m x o m x
| c 303 . 2 , =
III. Fluorescence Intensities
2a. Fluorescence spectra
Emission spectrum
Hold excitation wavelength fixed, scan emission
Reports on the fluorescence spectral profile
reflects fluorescence quantum yield, |
k
(
m
)

( ) ( ) ( )Z CL I F
m x o m x
| c 303 . 2 , =
III. Fluorescence Intensities
2b. Fluorescence spectra
Excitation spectrum
Hold emission wavelength fixed, scan excitation
Reports on absorption structure
reflects molar extinction coefficient, c(
x
)


( ) ( ) ( )Z CL I F
m x o m x
| c 303 . 2 , =
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

Emission Wavelength (nm)
Fixed Excitation Wavelength
(b)
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

Excitation Wavelength (nm)
Fixed Emission Wavelength
(a)
III. Fluorescence Intensities
III. Fluorescence Intensities
2c. Fluorescence spectra
Composite: Excitation-Emission Matrix
Good representation of multi-fluorophore solution

F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

Emission Wavelength (nm)
Fixed Excitation Wavelength
E
x
c
i
t
a
t
i
o
n

W
a
v
e
l
e
n
g
t
h

(
n
m
)

Emission Wavelength (nm)
Emission spectrum
Excitation-emission matrix
III. Fluorescence Intensities
IV. Biological Fluorophores
1. Table
2. EEMs of Epithelial cell suspension
3. EEMs of Collagen

IV. Biological Fluorophores
Endogenous Fluorophores
amino acids
structural proteins
enzymes and co-enzymes
vitamins
lipids
porphyrins
Exogenous Fluorophores
Cyanine dyes
Photosensitizers
Molecular markers GFP, etc.
IV. Biological Fluorophores
TCL-1
Emission(nm)
E
x
c
i
t
a
t
i
o
n
(
n
m
)

300 350 400 450 500 550 600 650 700
250
270
290
310
330
350
370
390
410
430
450
470
490
510
530
550
9.767e-001
6.646e+004
1.306e+005
1.947e+005
4.785e+005
1.120e+006
1.761e+006
2.923e+006
9.335e+006
1.575e+007
2.216e+007
Epithelial Cell Suspension
Fluorescence intensity excitation-emission
matrix
FAD
NADH
Tryp.
Courtesy of N. Ramanujam


Carbohydrates
Fatty Acids and Glycerol
Amino Acids
Acetyl CoA
CITRIC
ACID
CYCLE
CoA
CO
2
FADH
2

NADH
NADH-Q
Reductase
Cytochrome
Reductase
Cytochrome
Oxidase
Q
Cytochrome C
O
2
FAD NAD
+
Oxidation of NADH and FADH
2

by O
2
drives synthesis of ATP
ELECTRON
TRANSPORT
Metabolic Indicators

Metabolism

Redox Ratio: FAD / (FAD+NADH)

Redox ratio ~ Metabolic Rate




Emission(nm)
E
x
c
i
t
a
t
i
o
n
(
n
m
)
Highest value: 13223388.1 270/260
Scaled @: 4916595.85 305/270
M
S
UT Austin Mar-2000 UU
300 350 400 450 500 550 600 650 700
250
270
290
310
330
350
370
390
410
430
450
470
490
510
530
550
7.551e+003
2.210e+004
3.613e+004
5.016e+004
1.123e+005
2.526e+005
3.929e+005
6.473e+005
2.051e+006
3.454e+006
4.857e+006
0 days
Emission [nm]
E
x
c
i
t
a
t
i
o
n

[
n
m
]

Emission(nm)
E
x
c
i
t
a
t
i
o
n
(
n
m
)
Highest value: 9476234.1 270/260
Scaled @: 3424911.85 305/270
M
S
UT Austin Mar-2000 UU
300 350 400 450 500 550 600 650 700
250
270
290
310
330
350
370
390
410
430
450
470
490
510
530
550
5.021e+003
1.514e+004
2.490e+004
3.467e+004
7.788e+004
1.755e+005
2.731e+005
4.501e+005
1.426e+006
2.403e+006
3.379e+006
7 days
Collagen I (gel)
K. Sokolov
Emission(nm)
E
x
c
i
t
a
t
i
o
n
(
n
m
)
Highest value: 11516168.9 270/260
Scaled @: 4868431.748 305/270
M
S
UT Austin Mar-2000 UU
300 350 400 450 500 550 600 650 700
250
270
290
310
330
350
370
390
410
430
450
470
490
510
530
550
1.097e+004
2.559e+004
3.969e+004
5.379e+004
1.162e+005
2.572e+005
3.982e+005
6.538e+005
2.064e+006
3.474e+006
4.883e+006
39 days
IV. Biological Fluorophores
Collagen
It is the major extracellular matrix component, which is
present to some extent in nearly all organs and serves to
hold cells together in discrete units

Collagen fluorescence in load-bearing tissues is
associated with cross-links, hydroxylysyl pyridoline
(HP) and lysyl pyridinoline (LP).

Collagen crosslinks are altered with age and with
invasion of cancer into the extracellular matrix
V. Fluorescence Instrumentation
1. Introduction
2. Components of a spectrofluorometer
3. Description of key components


V. Fluorescence Instrumentation
1. Introduction
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
V. Fluorescence Instrumentation
Illumination source
Broadband (Xe lamp)
Monochromatic (LED, laser)
Light delivery to sample
Lenses/mirrors
Optical fibers
Wavelength separation (potentially for both excitation and
emission)
Monochromator
Spectrograph
Detector
PMT
CCD camera
2. Major components for fluorescence instrument
V. Fluorescence Instrumentation
Components of the spectrofluorometer (standard
fluorescence lab instrument for in vitro samples)
Xenon lamp (> 250 nm)

Excitation and emission monochromator
Each contains two gratings to increase purity of the light
Automatic scanning of wavelength through motorized gratings

Sample compartment

Photo multiplier tube
PMT
Xenon Source
Excitation
Monochromator
Emission
Monochromator
Sample compartment
V. Fluorescence Instrumentation
2. Spectrofluorometer schematic
V. Fluorescence Instrumentation
3a. Xenon light source
Continuous output from Xenon: 270-1100 nm

Power typically 200-450 W

Lifetime of ~2000 hours

Strong dependence on wavelength



V. Fluorescence Instrumentation
3a. Xenon light source: broad illumination in the
near UV-visible range


PMT
Xenon Source
Excitation
Monochromator
Emission
Monochromator
Sample compartment
V. Fluorescence Instrumentation
V. Fluorescence Instrumentation
3b. Monochromator: only a small range of wavelengths are focused
at the exit slit determined by angle of light incident on the
diffraction grating

constant
wavelength
mm per lines of # n
order n diffractio K where
, 10 sin sin
6
= =
=
=
=
= +

o |

| o
D
Kn
Principle of diffraction
grating operation
V. Fluorescence Instrumentation
3b. Monochromator Spectral Resolution
Inversely proportional to product of dispersion
(nm/mm) of grating and the slit width (mm)

~ 5 nm sufficient for fluorescence measurements of
biological media

Signal increases with the slit width

V. Fluorescence Instrumentation
3b. Monochromator Stray light
Light which passes through monochromator besides
that of desired wavelength

Double grating monochromator (stray light rejection
is 10
-8
10
-12
) but signal is decreased

V. Fluorescence Instrumentation
3b. Monochromator Signal efficiency
Grating has a wavelength dependent efficiency





Can choose the wavelength at which grating is blazed
(maximal efficiency)

Excitation monochromator should have high efficiency
in the UV; emission monochromator should have high
efficiency in the visible

PMT
Xenon Source
Excitation
Monochromator
Emission
Monochromator
Sample compartment
V. Fluorescence Instrumentation
V. Fluorescence Instrumentation
3c. Photomultiplier tube
Contains a photocathode:
light sensitive material, which
yields electrons upon
interaction with photons based
on photoelectric effect.
Electrons are multiplied by a
series of dynodes
Provides current output
proportional
to light intensity



V. Fluorescence Instrumentation
3c. PMT Linearity response
Current from PMT is proportional to light intensity

Under high intensity illumination, PMT will saturate
(dynamic range); at low intensity, limited by dark noise

Excessive light can damage photocathode, resulting in
loss of gain and increased dark noise (thermal noise)

V. Fluorescence Instrumentation
3c. PMT Quantum efficiency
Quantum efficiency gives the photon to electron
conversion efficiency

Highly dependent on wavelength


V. Fluorescence Instrumentation
3c. Key components Noise
Dark current Noise due to thermal generation;
increases with temperature and high voltage

Shot noise proportional to the square root of the
signal


VI. Fluorescence Measurements
VI. Fluorescence measurements
1. Instrument non-uniformities
2. Excitation wavelength calibration
3. Emission wavelength calibration
4. Setup parameters for emission spectrum
5. Routine experimental procedure
6. Collection geometry
7. Blank scans
8. Typical fluorescence spectrum
VI. Fluorescence Measurements
1a. Ideal spectrofluorometer
Light source must yield constant photon output at all
wavelengths

Monochromator must pass photons of all wavelengths
with equal efficiency

The PMT must detect photons of all wavelengths with
equal efficiency

VI. Fluorescence Measurements
L
i
g
h
t

I
n
t
e
n
s
i
t
y

Wavelength
LIGHT SOURCE
E
f
f
i
c
i
e
n
c
y

Wavelength
MONOCHROMATOR
E
f
f
i
c
i
e
n
c
y

Wavelength
PMT
VI. Fluorescence Measurements
1b. Distortions in excitation and emission spectra
Light intensity from light source is a function of
wavelength

Monochromator efficiency is a function of wavelength

The PMT does not have equal efficiency at all
wavelengths

VI. Fluorescence Measurements
1c. Calibration
Correction of variations in wavelength of Xenon lamp
and excitation monochromator
Need to do when measuring excitation spectra or emission
spectra at multiple excitation wavelengths

Correction of emission monochromator and PMT
Need to do when measuring emission spectra




VI. Fluorescence Measurements
2a. Excitation wavelength calibration
Excitation spectra are distorted primarily by the
wavelength dependent intensity of the light source

Can use reference photodetector (calibrated) next to
sample compartment to measure fraction of excitation
light

The measured intensity of the reference channel is
proportional to the intensity of the exciting light


I
o
F

Sample
Compartment
To PMT

To Reference Photodiode

VI. Fluorescence Measurements
2b. Effect of excitation wavelength calibration



VI. Fluorescence Measurements
3a. Emission wavelength calibration
Need correction factors
Measure wavelength dependent output from a
calibrated light source
Standard lamps of known and calibrated
spectral outputs are available from the National
Institute of Standards and Testing (NIST)
This measurement is typically done by factory;
it is difficult to perform properly with
commercial fluorimeter



To PMT

Sample
Compartment

Calibrated
Lamp

VI. Fluorescence Measurements
3b. Emission wavelength calibration procedure
Measure intensity versus wavelength (I()) of standard
lamp with spectrofluorometer

Obtain the spectral output data (L()) provided for the
lamp

Correction factor: S() = L()/ I()

Multiply emission spectrum with correction factor



VI. Fluorescence Measurements
3c. Emission wavelength calibration curve
0.0E+00
2.0E+00
4.0E+00
6.0E+00
8.0E+00
1.0E+01
1.2E+01
300 350 400 450 500 550 600 650 700
Wavelength (nm)
I
n
t
e
n
s
i
t
y

(
c
.
u
.
)
VI. Fluorescence Measurements
5. Routine experimental procedures
Check wavelength calibration of excitation
monochromator


Check wavelength calibration of emission
monochromator

Check throughput of spectrofluorometer




0.0E+00
1.0E-02
2.0E-02
3.0E-02
4.0E-02
5.0E-02
6.0E-02
260 310 360 410 460 510 560
Wavelength (nm)
I n
t e
n
s
i t y
(
c
p
s
)
467 nm
Xe lamp scan
Hg lamp spectrum scan
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1.0E+07
1.2E+07
475 525 575 625 675
Wavelength (nm)
I n
t e
n
s
i t y
( c
. u
. )
575 nm
Rhodamine standard
scan
VI. Fluorescence Measurements
6a. Collection geometry in sample compartment
Front face collection is at a 22 degree angle relative to
the incident beam; appropriate for an optically
absorbing / scattering sample; more stray light

Right angle collection is at a right angle to the
incident light; appropriate for optically transparent
sample; less stray light


I
o
F

I
o
F

Front Face Right Angle
VI. Fluorescence Measurements
6c. Features of right angle illumination
Appropriate for optically transparent sample

At high optical densities, signal reaching detector will
be significantly diminished


VI. Fluorescence Measurements
6d. Features of front face illumination
Appropriate for an optically absorbing / scattering
sample

At high optical densities, light is absorbed near the
surface of the cuvette containing the absorber; therefore
fluorescence is detectable

Fluorescence independent of concentration at high
optical densities


VI. Fluorescence Measurements
7. Blank scan
Blank is identical to sample except it does not contain
fluorophore

Measuring the fluorescence of these samples allows the
scattering (Rayleigh and Raman) to be assessed

In addition, such samples can reveal the presence of
fluorescence impurities, which can be subtracted

VI. Fluorescence Measurements
8. Typical fluorescence emission spectrum at 340 nm
excitation (the different components)

0
500000
1000000
1500000
2000000
2500000
3000000
300 350 400 450 500 550 600
Wavelength (nm)
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

(
a
.
u
.
)

Raman
Rayleigh (
exc
=
emm
)
Fluorescence