Lecture 15

Fluorescence spectroscopy and

imaging:
Basic principles and sources of
contrast
Outline for Fluorescence
I. Principles of Fluorescence
II. Quantum Yield and Lifetime
III. Fluorescence Spectroscopy
I. Biological Fluorop!ores
. Fluorescence Instrumentation
I. Fluorescence "easurements
I. Principles of Fluorescence
1. Luminescence
#
$mission of p!otons from electronically e%cited states
#
&'o types of luminescence:
(ela%ation from singlet e%cited state
(ela%ation from triplet e%cited state
I. Principles of Fluorescence
). Singlet and triplet states
#
*round state + t'o electrons per or,ital- electrons !a.e
opposite spin and are paired
#
Singlet e%cited state
$lectron in !ig!er energy or,ital !as t!e opposite spin
orientation relati.e to electron in t!e lo'er or,ital
#
&riplet e%cited state
&!e e%cited .alence electron may spontaneously re.erse its
spin /spin flip0. &!is process is called intersystem crossing.
$lectrons in ,ot! or,itals no' !a.e same spin orientation
I. Principles of Fluorescence
1. &ypes of emission
#
Fluorescence + return from e%cited singlet state to
ground state- does not re2uire c!ange in spin
orientation /more common of rela%ation0
#
P!osp!oresence + return from a triplet e%cited state to a
ground state- electron re2uires c!ange in spin
orientation
#
$missi.e rates of fluorescence are se.eral orders of
magnitude faster t!an t!at of p!osp!orescence
I. Principles of Fluorescence
3. $nergy le.el diagram /4a,lons5i diagram0
I. Principles of Fluorescence
5a. Fluorescence process: Population of energy le.els
#
6t room temperature /177 809 and for typical electronic
and .i,ration energy le.els9 can calculate t!e ratio of
molecules in upper and lo'er states
( )
kT
E
n
n
lower
upper

= e%p
5:1.1;<17
=)1
48
=1
/Bolt>mann?s constant0
$ : separation in energy le.el
I. Principles of Fluorescence
5,. Fluorescence process: $%citation
#
6t room temperature9 e.eryt!ing starts out at
t!e lo'est .i,rational energy le.el of t!e ground state
#
Suppose a molecule is illuminated 'it! lig!t at a
resonance fre2uency
#
Lig!t is a,sor,ed- for dilute sample9 Beer=Lam,ert
la' applies
'!ere is molar a,sorption /e%tinction0 coefficient
/"
=1
cm
=1
0- its magnitude reflects pro,a,ility of a,sorption and its 'a.elengt!
dependence corresponds to a,sorption spectrum
#
$%citation = follo'ing lig!t a,sorption9 a c!romop!ore is e%cited
to some higher .i,rational energy le.el of S
1
or S
)

#
&!e a,sorption process ta5es place on a time scale /17
=15
s0 muc! faster t!an
t!at of molecular .i,ration @ A.erticalB transition /Franc5=Condon
principle0.
So
S1
cl A =
E
n
e
r
g
y
nuclear configuration
I. Principles of Fluorescence
5c. Fluorescence process: Don=radiati.e rela%ation
#
In t!e e%cited state9 t!e electron is promoted
to an anti=,onding or,ital@ atoms in t!e ,ond
are less tig!tly !eld @ s!ift to t!e rig!t for S
1

potential energy cur.e @electron is promoted
to !ig!er .i,rational le.el in S
1
state t!an t!e
.i,rational le.el it 'as in at t!e ground state
#
i,rational deacti.ation ta5es place t!roug!
intermolecular collisions at a time scale of
17
=1)
s /faster t!an t!at of fluorescence
process0
.
So
S1
I. Principles of Fluorescence
5d. Fluorescence process: $mission
#
&!e molecule rela%es from t!e

lo'est .i,rational energy le.el
of t!e e%cited state to a .i,rational
energy le.el of t!e ground state
/17
=E
s0
#
(ela%ation to ground state occurs faster t!an
time scale of molecular .i,ration @ A.erticalB
transition

#
&!e energy of t!e emitted p!oton
is lo'er t!an t!at of t!e incident
p!otons
So
S1
I. Principles of Fluorescence
Fa. Sto5es s!ift
#
&!e fluorescence lig!t is red=s!ifted /longer 'a.elengt!
t!an t!e e%citation lig!t0 relati.e to t!e a,sor,ed lig!t
/GSto5es s!iftB0.
#
Internal con.ersion /see slide 110 can affect Sto5es s!ift
#
Sol.ent effects and e%cited state reactions can also affect
t!e magnitude of t!e Sto5e?s s!ift
I. Principles of Fluorescence
F,. In.ariance of emission 'a.elengt! 'it!
e%citation 'a.elengt!
#
$mission 'a.elengt! only
depends on rela%ation ,ac5
to lo'est .i,rational le.el of S
1
#
For a molecule9 t!e same
fluorescence emission 'a.elengt!
is o,ser.ed irrespecti.e of t!e
e%citation 'a.elengt!
So
S1
I. Principles of Fluorescence
Fc. "irror image rule
#
i,rational le.els in t!e e%cited states and
ground states are similar
#
6n a,sorption spectrum reflects t!e
.i,rational le.els of t!e electronically
e%cited state
#
6n emission spectrum reflects t!e
.i,rational le.els of t!e electronic ground
state
#
Fluorescence emission spectrum is mirror
image of a,sorption spectrum
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. Principles of Fluorescence
Fd. Internal con.ersion .s. fluorescence emission
# 6s electronic energy increases9 t!e energy le.els gro'
more closely spaced
# It is more li5ely t!at t!ere 'ill ,e o.erlap ,et'een t!e !ig!
.i,rational energy le.els of S
n=1
and lo' .i,rational energy
le.els of S
n


#
&!is o.erlap ma5es transition ,et'een states !ig!ly
pro,a,le
# Internal conversion is a transition occurring ,et'een
states of t!e same multiplicity and it ta5es place at a
time scale of 17
=1)
s /faster t!an t!at of fluorescence
process0
# &!e energy gap ,et'een S
1
and S
7
is significantly larger
t!an t!at ,et'een ot!er adHacent states @ S
1
lifetime is
longer @ radiati.e emission can compete effecti.ely 'it!
non=radiati.e emission
Mirror-image rule typically
applies !en only S
0
" S
1

e#citation ta$es place
%eviations from t!e mirror-
image rule are o&serve' !en
S
0
" S
2
or transitions to even
!ig!er e#cite' states also ta$e
place
I. Principles of fluorescence
Fe. Intersystem crossing
# Intersystem crossing refers to non=radiati.e transition ,et'een states of different
multiplicity
# It occurs .ia in.ersion of t!e spin of t!e e%cited electron resulting in t'o
unpaired electrons 'it! t!e same spin orientation9 resulting in a state 'it! Spin:1
and multiplicity of 1 /triplet state0
# &ransitions ,et'een states of different multiplicity are formally for,idden
# Spin=or,it and .i,ronic coupling mec!anisms decrease t!e ApureB c!aracter of
t!e initial and final states9 ma5ing intersystem crossing pro,a,le
# &
1
" S
7
transition is also for,idden " &
1
lifetime significantly larger t!an S
1

lifetime /17
=1
=17
)
s0
S
0
S
1
(
1
a&sorption
fluorescence
p!osp!orescence
Intersystem
crossing
I. Principles of fluorescence
I. Principles of fluorescence
I
n
t
e
n
s
i
t
y
)avelengt!
*&sor&ance
%+,+-
*&sor&ance
.luorescence .luorescence
*//E0(+-
Molecule 1 Molecule 2
#
Fluorescence energy transfer /F($&0
I
n
t
e
n
s
i
t
y
)avelengt!
*&sor&ance
%+,+-
*&sor&ance
.luorescence .luorescence
*//E0(+-
Molecule 1 Molecule 2
Non radiative energy transfer a quantum mechanical process of
resonance between transition dipoles
$ffecti.e ,et'een 17=177 I only
$mission and e%citation spectrum must significantly o.erlap
Jonor transfers non=radiati.ely to t!e acceptor
II. Quantum yield and lifetime
# Quantum yield of fluorescence9
f
9 is defined as:
#
In practice9 is measured ,y comparati.e measurements 'it! reference
compound for '!ic! !as ,een determined 'it! !ig! degree of accuracy.
#
Ideally9 reference compound s!ould !a.e
+ t!e same a,sor,ance as t!e compound of interest at gi.en e%citation 'a.elengt!
+ similar e%citation=emission c!aracteristics to compound of interest /ot!er'ise9
instrument 'a.elengt! response s!ould ,e ta5en into account0
+ Same sol.ent9 ,ecause intensity of emitted lig!t is dependent on refracti.e inde%
/ot!er'ise9 apply correction
+ Yields similar fluorescence intensity to ensure measurements are ta5en 'it!in t!e
range of linear instrument response
a,sor,ed p!otons of num,er
emitted p!otons of num,er
=
f
0 /
0 /
)
)
s n
u n
I
I
s
f
u
f
s
f
u
f
=

1a1 2uantum yiel' of fluorescence

II. Quantum yield and life time
# 6not!er definition for
f
is
'!ere k
r
is t!e radiati.e rate constant and k is t!e sum of t!e
rate constants for all processes t!at depopulate t!e S
1
state.
# In t!e a,sence of competing pat!'ays
f
:1
# (adiati.e lifetime9
r
9 is related to 5
r
#
&!e o,ser.ed fluorescence lifetime9 is t!e a.erage time t!e
molecule spends in t!e e%cited state9 and it is

=
k
k
r
f
r
r
k
1
=

=
k
f
1

1&1 .luorescence lifetime

II. Quantum Yield and Lifetime
)a. C!aracteristics of 2uantum yield
#
Quantum yield of fluorescence depends on ,iological
en.ironment
#
$%ample: Fura ) e%citation spectrum and Indo=1
emission spectrum and 2uantum yield c!ange '!en
,ound to Ca
)K
.ura-2 c!anges in response to
varying 3Ca)K4
In'o-1 c!anges in response to
varying 3Ca)K4
II. Quantum yield and lifetime
),. C!aracteristics of life=time
#
Pro.ide an additional dimension of information
missing in time=integrated steady=state spectral
measurements
#
Sensiti.e to ,ioc!emical microen.ironment9
including local pL9 o%ygenation and ,inding
#
Lifetimes unaffected ,y .ariations in e%citation
intensity9 concentration or sources of optical loss
#
Compati,le 'it! clinical measurements in .i.o
Courtesy of M.-A. Mycek, U Michigan
II. Quantum Yield and Lifetime
1a. Fluorescence emission distri,ution
#
For a gi.en e%citation 'a.elengt!9
t!e emission transition is
distri,uted among different
.i,rational energy le.els
#
For a single e%citation 'a.elengt!9
can measure a fluorescence
emission spectrum
I
n
t
e
n
s
i
t
y
$mission Ma.elengt! /nm0
E#c
Emm
II. Quantum Yield and Lifetime
1,. Leisen,erg?s uncertainty principle
#
alues of particular pairs of o,ser.a,les cannot ,e
determined simultaneously 'it! !ig! precision in
2uantum mec!anics
#
$%ample of pairs of o,ser.a,les t!at are restricted in
t!is 'ay are:
#
"omentum and position
#
$nergy and time
II. Quantum Yield and Lifetime
1c. Leisen,erg?s uncertainty principle
#
"omentum and position :
#
$nergy and time:
)
!
# p
#

)
!
t E
II. Quantum Yield and Lifetime
1d. $ffect on fluorescence emission
#
Suppose an e%cited molecule emits fluorescence in
rela%ing ,ac5 to t!e ground state
#
If t!e e%cited state lifetime9 is long9 t!en emission
'ill ,e monoc!romatic /single line0
#
If t!e e%cited state lifetime9 is s!ort9 t!en emission
'ill !a.e a 'ider range of fre2uencies /multiple lines0
I
n
t
e
n
s
i
t
y
$mission Ma.elengt! /nm0
E#c
Emm
I
n
t
e
n
s
i
t
y
$mission Ma.elengt! /nm0
E#c Emm
Large + small
Small + large
III. Fluorescence Intensity
1. Fluorescence intensity e%pression
). Fluorescence spectra
III. Fluorescence Intensities
1a. Fluorescence intensity
&!e fluorescence intensity /F0 at a particular e%citation
/
%
0 and emission 'a.elengt! /
m
0 'ill depend on t!e
a,sorption and t!e 2uantum yield:
'!ere9
I
6
+ lig!t a,sor,ed to promote electronic transition
+ 2uantum yield
( ) ( ) ( )
m # * m #
I . = 9
III. Fluorescence Intensities
1,. From t!e Beer=Lam,ert la'9 t!e a,sor,ed intensity
for a dilute solution /.ery small a,sor,ance0
'!ere9
I
o
+ Initial intensity
+ molar e%tinction coefficient
C + concentration
L + pat! lengt!
1 55 /6 for
/6 I 303 1 2 I
#
# o # *
III. Fluorescence Intensities
1c. Fluorescence intensity e%pression
&!e fluorescence intensity /F0 at a particular e%citation
/
%
0 and emission 'a.elengt! /
m
0 for a dilute solution
containing a fluorop!ore is:
'!ere9
I
o
+ incident lig!t intensity + 2uantum yield
C + concentration + molar e%tinction
L + pat! lengt! coefficient
( ) ( ) ( )
m # o m #
/6 I . 171 . ) 9 =
III. Fluorescence Intensities
1d. "easured fluorescence intensity
If 'e include instrument collection angle:
'!ere9
N + instrumental factor
I
o
+ incident lig!t intensity
+ molar e%tinction coefficient
C : concentration
L + pat! lengt!
( ) ( ) ( ) 7 /6 I .
m # o m #
171 . ) 9 =
III. Fluorescence Intensities
)a. Fluorescence spectra
#
$mission spectrum
+
Lold e%citation 'a.elengt! fi%ed9 scan emission
+
(eports on t!e fluorescence spectral profile
O reflects fluorescence 2uantum yield,
k
(
m
)
( ) ( ) ( ) 7 /6 I .
m # o m #
171 . ) 9 =
III. Fluorescence Intensities
),. Fluorescence spectra
#
$%citation spectrum
+
Lold emission 'a.elengt! fi%ed9 scan e%citation
+
(eports on a,sorption structure
O reflects molar e%tinction coefficient9 /
%
0
( ) ( ) ( ) 7 /6 I .
m # o m #
171 . ) 9 =
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y
$mission Ma.elengt! /nm0
Fi%ed $%citation Ma.elengt!
/,0
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y
$%citation Ma.elengt! /nm0
Fi%ed $mission Ma.elengt!
/a0
III. Fluorescence Intensities
III. Fluorescence Intensities
)c. Fluorescence spectra
#
Composite: $%citation=$mission "atri%
O
*ood representation of multi=fluorop!ore solution
.
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y
Emission )avelengt! 8nm9
.i#e' E#citation )avelengt!
E
#
c
i
t
a
t
i
o
n

)
a
v
e
l
e
n
g
t
!

8
n
m
9
Emission )avelengt! 8nm9
$mission spectrum
$%citation=emission matri%
III. Fluorescence Intensities
I. Biological Fluorop!ores
1. &a,le
). $$"s of $pit!elial cell suspension
1. $$"s of Collagen
I. Biological Fluorop!ores
+
$ndogenous Fluorop!ores
amino acids
structural proteins
en>ymes and co=en>ymes
.itamins
lipids
porp!yrins
+
$%ogenous Fluorop!ores
Cyanine dyes
P!otosensiti>ers
"olecular mar5ers + *FP9 etc.
I. Biological Fluorop!ores
&CL=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
$pit!elial Cell Suspension
Fluorescence intensity e%citation=emission
matri%
FAD
NADH
Tryp.
/ourtesy of ,1 -amanu:am
/ar&o!y'rates
.atty *ci's an' ;lycerol
*mino *ci's
*cetyl /o*
/I(-I/
*/I%
/</6E
/o*
/+
2
.*%=
2
,*%=
,*%=-2
-e'uctase
/ytoc!rome
-e'uctase
/ytoc!rome
+#i'ase
2
/ytoc!rome /
+
2
.*% ,*%
>
+#i'ation of ,*%= an' .*%=
2

&y +
2
'rives synt!esis of *(0
E6E/(-+,
(-*,S0+-(
"eta,olic Indicators
"eta,olism
-e'o# -atio? .*% @ 8.*%>,*%=9

-e'o# ratio P Meta&olic -ate
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 Aust in 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
7 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 Aust in 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
Q days
Collagen I /gel0
8. So5olo.
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 Aust in 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
1E days
I. Biological Fluorop!ores
Collagen
#
It is t!e maHor e%tracellular matri% component9 '!ic! is
present to some e%tent in nearly all organs and ser.es to
!old cells toget!er in discrete units
#
Collagen fluorescence in load=,earing tissues is
associated 'it! cross=lin5s9 !ydro%ylysyl pyridoline
/LP0 and lysyl pyridinoline /LP0.
#
Collagen crosslin5s are altered 'it! age and 'it!
in.asion of cancer into t!e e%tracellular matri%
. Fluorescence Instrumentation
1. Introduction
). Components of a spectrofluorometer
1. Jescription of 5ey components
. Fluorescence Instrumentation
1. Introduction
#
Fluorescence is a !ig!ly sensiti.e met!od /can measure
analyte concentration of 17
=;
"0
#
Important to minimi>e interference from:
Bac5ground fluorescence from sol.ents
Lig!t lea5s in t!e instrument
Stray lig!t scattered ,y tur,id solutions
#
Instruments do not yield ideal spectra:
Don=uniform spectral output of lig!t source
Ma.elengt! dependent efficiency of detector and
optical elemens
. Fluorescence Instrumentation
#
Illumination source
+
Broad,and /Re lamp0
+
"onoc!romatic /L$J9 laser0
#
Lig!t deli.ery to sample
+
LensesSmirrors
+
Optical fi,ers
#
Ma.elengt! separation /potentially for ,ot! e%citation and
emission0
+
"onoc!romator
+
Spectrograp!
#
Jetector
+
P"&
+
CCJ camera
21 Ma:or components for fluorescence instrument
. Fluorescence Instrumentation
Components of t!e spectrofluorometer /standard
fluorescence la, instrument for in .itro samples0
#
Renon lamp /T )57 nm0
#
$%citation and emission monoc!romator
$ac! contains t'o gratings to increase purity of t!e lig!t
6utomatic scanning of 'a.elengt! t!roug! motori>ed gratings
#
Sample compartment
#
P!oto multiplier tu,e
P"&
Renon Source
$%citation
"onoc!romator
$mission
"onoc!romator
Sample compartment
. Fluorescence Instrumentation
21 Spectrofluorometer sc!ematic
. Fluorescence Instrumentation
1a. Renon lig!t source
#
Continuous output from Renon: )Q7=1177 nm
#
Po'er + typically )77=357 M
#
Lifetime of P)777 !ours
#
Strong dependence on 'a.elengt!
. Fluorescence Instrumentation
1a. Renon lig!t source: ,road illumination in t!e
near U=.isi,le range
P"&
Renon Source
$%citation
"onoc!romator
$mission
"onoc!romator
Sample compartment
. Fluorescence Instrumentation
. Fluorescence Instrumentation
1,. "onoc!romator: only a small range of 'a.elengt!s are focused
at t!e e%it slit determined ,y angle of lig!t incident on t!e
diffraction grating
constant
'a.elengt!
mm per lines of V n
order n diffractio 8 '!ere
9 17 sin sin
F
= =
=
=
=
= +

D
n
0rinciple of 'iffraction
grating operation
. Fluorescence Instrumentation
1,. "onoc!romator + Spectral (esolution
In.ersely proportional to product of dispersion
/nmSmm0 of grating and t!e slit 'idt! /mm0
P 5 nm sufficient for fluorescence measurements of
,iological media
Signal increases 'it! t!e slit 'idt!
. Fluorescence Instrumentation
1,. "onoc!romator + Stray lig!t
#
Lig!t '!ic! passes t!roug! monoc!romator ,esides
t!at of desired 'a.elengt!
#
Jou,le grating monoc!romator /stray lig!t reHection
is 17
=;
+ 17
=1)
0 ,ut signal is decreased
. Fluorescence Instrumentation
1,. "onoc!romator + Signal efficiency
#
*rating !as a 'a.elengt! dependent efficiency
#
Can c!oose t!e 'a.elengt! at '!ic! grating is ,la>ed
/ma%imal efficiency0
#
$%citation monoc!romator s!ould !a.e !ig! efficiency
in t!e U- emission monoc!romator s!ould !a.e !ig!
efficiency in t!e .isi,le
P"&
Renon Source
$%citation
"onoc!romator
$mission
"onoc!romator
Sample compartment
. Fluorescence Instrumentation
. Fluorescence Instrumentation
1c. P!otomultiplier tu,e
#
Contains a p!otocat!ode:
lig!t sensiti.e material9 '!ic!
yields electrons upon
interaction 'it! p!otons ,ased
on p!otoelectric effect.
#
$lectrons are multiplied ,y a
series of dynodes
#
Pro.ides current output
proportional
to lig!t intensity
. Fluorescence Instrumentation
1c. P"& + Linearity response
#
Current from P"& is proportional to lig!t intensity
#
Under !ig! intensity illumination9 P"& 'ill saturate
/dynamic range0- at lo' intensity9 limited ,y dar5 noise
#
$%cessi.e lig!t can damage p!otocat!ode9 resulting in
loss of gain and increased dar5 noise /t!ermal noise0
. Fluorescence Instrumentation
1c. P"& + Quantum efficiency
#
Quantum efficiency gi.es t!e p!oton to electron
con.ersion efficiency
#
Lig!ly dependent on 'a.elengt!
. Fluorescence Instrumentation
1c. 8ey components + Doise
#
Jar5 current + Doise due to t!ermal generation-
increases 'it! temperature and !ig! .oltage
#
S!ot noise + proportional to t!e s2uare root of t!e
signal
I. Fluorescence "easurements
I. Fluorescence measurements
1. Instrument non=uniformities
). $%citation 'a.elengt! cali,ration
1. $mission 'a.elengt! cali,ration
3. Setup parameters for emission spectrum
5. (outine e%perimental procedure
F. Collection geometry
Q. Blan5 scans
;. &ypical fluorescence spectrum
I. Fluorescence "easurements
1a. Ideal spectrofluorometer
#
Lig!t source must yield constant p!oton output at all
'a.elengt!s
#
"onoc!romator must pass p!otons of all 'a.elengt!s
'it! e2ual efficiency
#
&!e P"& must detect p!otons of all 'a.elengt!s 'it!
e2ual efficiency
I. Fluorescence "easurements
L
i
g
!
t

I
n
t
e
n
s
i
t
y
Ma.elengt!
LI*L& SOU(C$
$
f
f
i
c
i
e
n
c
y
Ma.elengt!
"ODOCL(O"6&O(
$
f
f
i
c
i
e
n
c
y
Ma.elengt!
P"&
I. Fluorescence "easurements
1,. Jistortions in e%citation and emission spectra
#
Lig!t intensity from lig!t source is a function of
'a.elengt!
#
"onoc!romator efficiency is a function of 'a.elengt!
#
&!e P"& does not !a.e e2ual efficiency at all
'a.elengt!s
I. Fluorescence "easurements
1c. Cali,ration
#
Correction of .ariations in 'a.elengt! of Renon lamp
and e%citation monoc!romator
Deed to do '!en measuring e%citation spectra or emission
spectra at multiple e%citation 'a.elengt!s
#
Correction of emission monoc!romator and P"&
Deed to do '!en measuring emission spectra
I. Fluorescence "easurements
)a. $%citation 'a.elengt! cali,ration
#
$%citation spectra are distorted primarily ,y t!e
'a.elengt! dependent intensity of t!e lig!t source
#
Can use reference p!otodetector /cali,rated0 ne%t to
sample compartment to measure fraction of e%citation
lig!t
#
&!e measured intensity of t!e reference c!annel is
proportional to t!e intensity of t!e e%citing lig!t
I
o
F
Sample
Compartment
&o P"&
&o (eference P!otodiode
I. Fluorescence "easurements
),. $ffect of e%citation 'a.elengt! cali,ration
I. Fluorescence "easurements
1a. $mission 'a.elengt! cali,ration
#
Deed correction factors
#
"easure 'a.elengt! dependent output from a
cali,rated lig!t source
#
Standard lamps of 5no'n and cali,rated
spectral outputs are a.aila,le from t!e Dational
Institute of Standards and &esting /DIS&0
#
&!is measurement is typically done ,y factory-
it is difficult to perform properly 'it!
commercial fluorimeter
&o P"&
Sample
Compartment
Cali,rated
Lamp
I. Fluorescence "easurements
1,. $mission 'a.elengt! cali,ration procedure
#
"easure intensity .ersus 'a.elengt! /I/00 of standard
lamp 'it! spectrofluorometer
#
O,tain t!e spectral output data /L/00 pro.ided for t!e
lamp
#
Correction factor: S/0 : L/0S I/0
#
"ultiply emission spectrum 'it! correction factor
I. Fluorescence "easurements
1c. $mission 'a.elengt! cali,ration cur.e
0.0E+00
2.0E+00
.0E+00
!.0E+00
".0E+00
#.0E+0#
#.2E+0#
$00 $%0 00 %0 %00 %%0 !00 !%0 &00
'a(e)ength *nm+
,
n
t
e
n
s
i
t
y

*
c
.
u
.
+
I. Fluorescence "easurements
5. (outine e%perimental procedures
#
C!ec5 'a.elengt! cali,ration of e%citation
monoc!romator
#
C!ec5 'a.elengt! cali,ration of emission
monoc!romator
#
C!ec5 t!roug!put of spectrofluorometer
0.0E+00
#.0E-02
2.0E-02
$.0E-02
.0E-02
%.0E-02
!.0E-02
2!0 $#0 $!0 #0 !0 %#0 %!0
'a(e)ength *nm+
,n
te
n
s
ity
*
c
-
s
+
467 nm
Ae lamp scan
=g lamp spectrum scan
0.0E+00
2.0E+0!
.0E+0!
!.0E+0!
".0E+0!
#.0E+0&
#.2E+0&
&% %2% %&% !2% !&%
'a(e)ength *nm+
,n
te
n
s
ity
*c
.u
.+
575 nm
-!o'amine stan'ar'
scan
I. Fluorescence "easurements
Fa. Collection geometry in sample compartment
#
Front face + collection is at a )) degree angle relati.e
to t!e incident ,eam- appropriate for an optically
a,sor,ing S scattering sample- more stray lig!t
#
(ig!t angle + collection is at a rig!t angle to t!e
incident lig!t- appropriate for optically transparent
sample- less stray lig!t
I
o
F
I
o
F
Front Face (ig!t 6ngle
I. Fluorescence "easurements
Fc. Features of rig!t angle illumination
#
6ppropriate for optically transparent sample
#
6t !ig! optical densities9 signal reac!ing detector 'ill
,e significantly diminis!ed
I. Fluorescence "easurements
Fd. Features of front face illumination
#
6ppropriate for an optically a,sor,ing S scattering
sample
#
6t !ig! optical densities9 lig!t is a,sor,ed near t!e
surface of t!e cu.ette containing t!e a,sor,er- t!erefore
fluorescence is detecta,le
#
Fluorescence independent of concentration at !ig!
optical densities
I. Fluorescence "easurements
Q. Blan5 scan
#
Blan5 is identical to sample e%cept it does not contain
fluorop!ore
#
"easuring t!e fluorescence of t!ese samples allo's t!e
scattering /(ayleig! and (aman0 to ,e assessed
#
In addition9 suc! samples can re.eal t!e presence of
fluorescence impurities9 '!ic! can ,e su,tracted
I. Fluorescence "easurements
;. &ypical fluorescence emission spectrum at 137 nm
e%citation /t!e different components0
0
%00000
#000000
#%00000
2000000
2%00000
$000000
$00 $%0 00 %0 %00 %%0 !00
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
.
)
(aman
(ayleig! /
e%c
:
emm
0
Fluorescence