Introduction Fluorescence

8/12/2019 Introduction Fluorescence

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Lecture 15

Fluorescence spectroscopy andimaging:

Basic principles and sources ofcontrast


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Outline for Fluorescence

I. Principles of Fluorescence

II. Quantum Yield and Lifetime

III. Fluorescence SpectroscopyI. Biological Fluorop!ores

. Fluorescence Instrumentation

I. Fluorescence "easurements


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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


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I. Principles of Fluorescence). Singlet and triplet states

# *round state + t'o electrons per or,ital- electrons !aeopposite spin and are paired

# Singlet e%cited state$lectron in !ig!er energy or,ital !as t!e opposite spinorientation relatie to electron in t!e lo'er or,ital

# &riplet e%cited state&!e e%cited alence electron may spontaneously reerse itsspin /spin flip0. &!is process is called intersystem crossing.$lectrons in ,ot! or,itals no' !ae same spin orientation


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.&ypes of emission# Fluorescence + return from e%cited singlet state to

ground state- does not re2uire c!ange in spinorientation /more common of rela%ation0

# P!osp!oresence + return from a triplet e%cited state to aground state- electron re2uires c!ange in spinorientation

# $missie rates of fluorescence are seeral orders ofmagnitude faster t!an t!at of p!osp!orescence


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I. Principles of Fluorescence3.$nergy leel diagram /4a,lonsi diagram0


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5a. Fluorescence process: Population of energy leels# 6t room temperature /77 809 and for typical electronic

and i,ration energy leels9 can calculate t!e ratio of

molecules in upper and lo'er states

( )kT

En

n

lower

upper = e%p

1.;mann?s constant0$ separation in energy leel


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I. Principles of Fluorescence5,. Fluorescence process: $%citation

# 6t room temperature9 eeryt!ing starts out att!e lo'est i,rational energy leel of t!e ground state

# Suppose a molecule is illuminated 'it! lig!t at aresonance fre2uency

# Lig!t is a,sor,ed- for dilute sample9 Beer=Lam,ertla' applies'!ere is molar a,sorption /e%tinction0 coefficient/"=1cm=10- its magnitude reflects pro,a,ility of a,sorption and its 'aelengt!dependence corresponds to a,sorption spectrum

# $%citation = follo'ing lig!t a,sorption9 a c!romop!ore is e%citedto some higheri,rational energy leel of S

1or S

)

# &!e a,sorption process taes place on a time scale /17 =15s0 muc! faster t!an

t!at of molecular i,ration@ Aertical transition /Franc=Condonprinciple0.

So

S1

clA =

Energy

nuclear configuration


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I. Principles of Fluorescence5c. Fluorescence process: Don=radiatie rela%ation

# In t!e e%cited state9 t!e electron is promotedto an anti=,onding or,ital@ atoms in t!e ,ondare less tig!tly !eld @ s!ift to t!e rig!t for S

1

potential energy cure @electron is promotedto !ig!er i,rational leel in S1state t!an t!ei,rational leel it 'as in at t!e ground state

# i,rational deactiation taes place t!roug!

intermolecular collisions at a time scale of17=1)s /faster t!an t!at of fluorescenceprocess0

.

So

S1


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I. Principles of Fluorescence5d. Fluorescence process: $mission

# &!e molecule rela%es from t!e

lo'est i,rational energy leel

of t!e e%cited state to a i,rationalenergy leel of t!e ground state/17=Es0

#(ela%ation to ground state occurs faster t!antime scale of molecular i,ration @ Aerticaltransition

# &!e energy of t!e emitted p!oton

is lo'er t!an t!at of t!e incidentSo

S1


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a.Stoes s!ift# &!e fluorescence lig!t is red=s!ifted /longer 'aelengt!

t!an t!e e%citation lig!t0 relatie to t!e a,sor,ed lig!t/GStoes s!ift0.

# Internal conersion /see slide 10 can affect Stoes s!ift

# Solent effects and e%cited state reactions can also affect

t!e magnitude of t!e Stoe?s s!ift


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,. Inariance of emission 'aelengt! 'it!e%citation 'aelengt!

# $mission 'aelengt! only

depends on rela%ation ,acto lo'est i,rational leel of S

1

# For a molecule9 t!e same

fluorescence emission 'aelengt!

is o,sered irrespectie of t!e

e%citation 'aelengt!

So

S1


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I. Principles of Fluorescencec. "irror image rule

# i,rational leels in t!e e%cited states andground states are similar

# 6n a,sorption spectrum reflects t!e

i,rational leels of t!e electronicallye%cited state

# 6n emission spectrum reflects t!e

i,rational leels of t!e electronic groundstate

# Fluorescence emission spectrum is mirrorimage of a,sorption spectrum

S0

S1

v=0

v=1

v=2v=3

v=4v=5

v=0

v=1v=2

v=3v=4v=5


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I. Principles of Fluorescenced. Internal conersion s. fluorescence emission

# 6s electronic energy increases9 t!e energy leels gro'more closely spaced

# It is more liely t!at t!ere 'ill ,e oerlap ,et'een t!e !ig!i,rational energy leels of S

n=1and lo' i,rational energy

leels of Sn

# &!is oerlap maes transition ,et'een states !ig!lypro,a,le

# Internal conversionis a transition occurring ,et'eenstates of t!e same multiplicity and it taes place at atime scale of 17=1)s /faster t!an t!at of fluorescence

process0# &!e energy gap ,et'een S

1and S

7is significantly larger

t!an t!at ,et'een ot!er adHacent states @ S1lifetime is

longer @ radiatie emission can compete effectiely 'it!non=radiatie emission


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Mirror-image rule typically

applies !en only S0" S1

e#citation ta$es place

%eviations from t!e mirror-image rule are o&serve' !en

S0" S2or transitions to even

!ig!er e#cite' states also ta$e

place


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I. Principles of fluorescencee. Intersystem crossing# Intersystem crossing refers to non=radiatie transition ,et'een states of different

multiplicity

# It occurs ia inersion of t!e spin of t!e e%cited electron resulting in t'ounpaired electrons 'it! t!e same spin orientation9 resulting in a state 'it! Spin1and multiplicity of /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 Apure c!aracter oft!e initial and final states9 maing intersystem crossing pro,a,le

# &1"S

7transition is also for,idden "&

1lifetime significantly larger t!an S

1

lifetime /17==17)s0

S0

S1(1

a&sorptionfluorescence

p!osp!orescence

Intersystem

crossing


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I. Principles of fluorescence

Intensity

)avelengt!

*&sor&ance

%+,+

*&sor&ance

.luorescence .luorescence

*//E(+

Molecule 1 Molecule 2

# Fluorescence energy transfer /F($&0

Intensity

)avelengt!

*&sor&ance

%+,+

*&sor&ance

.luorescence .luorescence

*//E(+

Molecule 1 Molecule 2

Non radiative energy transfer a quantum mechanical process of

resonance between transition dipoles

$ffectie ,et'een 17=177 only$mission and e%citation spectrum must significantly oerlap

Jonor transfers non=radiatielyto t!e acceptor


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II. Quantum yield and lifetime

# Quantum yield of fluorescence9 f9 is defined as:

# In practice9 is measured ,y comparatie measurements 'it! referencecompound for '!ic! !as ,een determined 'it! !ig! degree of accuracy.

# Ideally9 reference compound s!ould !ae+

t!e same a,sor,ance as t!e compound of interest at gien e%citation 'aelengt!+ similar e%citation=emission c!aracteristics to compound of interest /ot!er'ise9instrument 'aelengt! response s!ould ,e taen into account0

+ Same solent9 ,ecause intensity of emitted lig!t is dependent on refractie inde%/ot!er'ise9 apply correction

a,sor,edp!otonsofnum,er

emittedp!otonsofnum,er= f

0/

0/)

)

sn

un

I

Is

f

u

f

s

f

u

f=

1a uantum yiel' of fluorescence


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II. Quantum yield and life time

# 6not!er definition for fis

'!ere kr

is t!e radiatie rate constant and kis t!e sum of t!erate constants for all processes t!at depopulate t!e S

1state.

# In t!e a,sence of competing pat!'ays f1

# (adiatie lifetime9 r9 is related to

r

# &!e o,sered fluorescence lifetime9 is t!e aerage time t!emolecule spends in t!e e%cited state9 and it is

=

k

krf

r

rk

1=

=

kf

1

1& .luorescence lifetime


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)a. C!aracteristics of 2uantum yield# Quantum yield of fluorescence depends on ,iological

enironment

# $%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 Ca)K

In'o-1 c!anges in response to

varying Ca)K


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a. Fluorescence emission distri,ution# For a gien e%citation 'aelengt!9

t!e emission transition is

distri,uted among differenti,rational energy leels

# For a single e%citation 'aelengt!9

can measure a fluorescenceemission spectrum

Intensity

$mission Maelengt! /nm0

E#cEmm


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,. eisen,erg?s uncertainty principle# alues of particular pairs of o,sera,les cannot ,e

determined simultaneously 'it! !ig! precision in

2uantum mec!anics

# $%ample of pairs of o,sera,les t!at are restricted int!is 'ay are:

# "omentum and position

# $nergy and time


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c. eisen,erg?s uncertainty principle

# "omentum and position:

# $nergy and time:

)

!#p

#

)

!tE


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d. $ffect on fluorescence emission# Suppose an e%cited molecule emits fluorescence in

rela%ing ,ac 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 !ae a 'ider range of fre2uencies /multiple lines0


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Intensit

y


E#c Emm

Intensi

ty


E#c Emm

Large

+ small Small + large


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III. Fluorescence Intensity

1. Fluorescence intensity e%pression

). Fluorescence spectra


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III. Fluorescence Intensities

1a. Fluorescence intensity&!e fluorescence intensity /F0 at a particular e%citation/

%0 and emission 'aelengt! /

m0 'ill depend on t!e

a,sorption and t!e 2uantum yield:

'!ere9

I6+ lig!t a,sor,ed to promote electronic transition

+ 2uantum yield

( ) ( ) ( )m#*m# I. =9


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1,. From t!e Beer=Lam,ert la'9 t!e a,sor,ed intensityfor a dilute solution /ery small a,sor,ance0

'!ere9

Io+ Initial intensity+ molar e%tinction coefficient

C + concentrationL + pat! lengt!

155/6for

/6I3032I

#

#o#*


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1c. Fluorescence intensity e%pression&!e fluorescence intensity /F0 at a particular e%citation/

%0 and emission 'aelengt! /

m0 for a dilute solution

containing a fluorop!ore is:

'!ere9

Io+ incident lig!t intensity + 2uantum yield

C + concentration + molar e%tinction

L + pat! lengt! coefficient

( ) ( ) ( )m#om# /6I. 171.)9 =


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1d. "easured fluorescence intensityIf 'e include instrument collection angle:

'!ere9

N + instrumental factor

Io+ incident lig!t intensity+ molar e%tinction coefficient

C concentration

L + pat! lengt!

( ) ( ) ( )7/6I. m#om# 171.)9 =


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)a. Fluorescence spectra# $mission spectrum

+ old e%citation 'aelengt! fi%ed9 scan emission

+ (eports on t!e fluorescence spectral profile

reflects fluorescence 2uantum yield, k(m)

( ) ( ) ( )7/6I. m#om# 171.)9 =


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),. Fluorescence spectra# $%citation spectrum

+ old emission 'aelengt! fi%ed9 scan e%citation

+ (eports on a,sorption structure

reflects molar e%tinction coefficient9 /%0

( ) ( ) ( )7/6I.m#om#

171.)9 =


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Fluorescence

Intensity


Fi%ed $%citation Maelengt!

/,0

Fluorescence

Intensity

$%citation Maelengt! /nm0

Fi%ed $mission Maelengt!

/a0



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)c. Fluorescence spectra# Composite: $%citation=$mission "atri%

*ood representation of multi=fluorop!ore solution


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I. Biological Fluorop!ores

1. &a,le

). $$"s of $pit!elial cell suspension

. $$"s of Collagen


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I. Biological Fluorop!ores+$ndogenous Fluorop!ores

amino acids

structural proteins

en>ymes and co=en>ymes

itaminslipids

porp!yrins

+$%ogenous Fluorop!ores

Cyanine dyes

P!otosensiti>ers

"olecular marers + *FP9 etc.


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/ar&o!y'rates

.atty *ci's an' ;lycerol

*mino *ci's

*cetyl /o*

/I(I/ */I%

/


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"eta,olic Indicators

"eta,olism

e'o# atio? .*% @ 8.*%>,*%9

e'o# ratio Meta&olic ate


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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 70025 0

27 0

29 031 0

33 0

35 0

37 0

39 0

41 0

43 0

45 0

47 0

49 0

51 053 0

55 0

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]

Excitation

[nm]

Highest value: 9476234 .1 270/260

Scaled @: 3424911.85 305/270

M

S


300 350 400 450 500 550 600 650 70025 0

27 0

29 031 0

33 0

35 0

37 0

39 0

41 0

43 0

45 0

47 0

49 0

51 053 0

55 0

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

days

Collagen I /gel0

8. Soolo

Highest value: 11516168 .9 270/260

Scaled @: 4868431.748 305/270

M

S


300 350 400 450 500 550 600 650 70025 0

27 0

29 031 0

33 0

35 0

37 0

39 0

41 0

43 0

45 0

47 0

49 0

51 053 0

55 0

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

E days


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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 seres to!old cells toget!er in discrete units

# Collagen fluorescence in load=,earing tissues isassociated 'it! cross=lins9 !ydro%ylysyl pyridoline/P0 and lysyl pyridinoline /LP0.

# Collagen crosslins are altered 'it! age and 'it!inasion of cancer into t!e e%tracellular matri%


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1. Introduction

). Components of a spectrofluorometer

. Jescription of ey components


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1. Introduction# Fluorescence is a !ig!ly sensitie met!od /can measure

analyte concentration of 17=;"0

# Important to minimi>e interference from:Bacground fluorescence from solents

Lig!t leas 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

Maelengt! dependent efficiency of detector andoptical elemens


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# Illumination source+ Broad,and /Re lamp0+ "onoc!romatic /L$J9 laser0

# Lig!t deliery to sample+

Lensesmirrors+ Optical fi,ers

# Maelengt! separation /potentially for ,ot! e%citation andemission0+ "onoc!romator+ Spectrograp!

# Jetector+ P"&+ CCJ camera

2 Ma:or components for fluorescence instrument


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Components of t!e spectrofluorometer /standardfluorescence 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!t6utomatic scanning of 'aelengt! t!roug! motori>ed gratings

# Sample compartment

# P!oto multiplier tu,e


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a. Renon lig!t source# Continuous output from Renon: )7=1177 nm

#Po'er + typically )77=357 M

# Lifetime of )777 !ours

# Strong dependence on 'aelengt!


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a. Renon lig!t source: ,road illumination in t!enear U=isi,le range


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P"&

Renon Source

$%citation"onoc!romator $mission"onoc!romator

Sample compartment


Fl I i


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. Fluorescence Instrumentation,. "onoc!romator: only a small range of 'aelengt!s are focused

at t!e e%it slit determined ,y angle of lig!t incident on t!ediffraction grating

constant

'a.elengt!

mmperlinesofVn

orderndiffractio8'!ere

917sinsin F

==

=

=

=

=+

D

n

rinciple of 'iffraction

grating operation


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,. "onoc!romator + Spectral (esolutionInersely proportional to product of dispersion

/nmmm0 of grating and t!e slit 'idt! /mm0

5 nm sufficient for fluorescence measurements of

,iological media

Signal increases 'it! t!e slit 'idt!


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,. "onoc!romator + Stray lig!t# Lig!t '!ic! passes t!roug! monoc!romator ,esides

t!at of desired 'aelengt!

# Jou,le grating monoc!romator /stray lig!t reHectionis 17 =;+ 17=1)0 ,ut signal is decreased


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,. "onoc!romator + Signal efficiency# *rating !as a 'aelengt! dependent efficiency

# Can c!oose t!e 'aelengt! at '!ic! grating is ,la>ed/ma%imal efficiency0

# $%citation monoc!romator s!ould !ae !ig! efficiencyin t!e U- emission monoc!romator s!ould !ae !ig!efficiency in t!e isi,le


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P"&

Renon Source

$%citation"onoc!romator $mission"onoc!romator

Sample compartment



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c. P!otomultiplier tu,e# Contains a p!otocat!ode:

lig!t sensitie material9 '!ic!

yields electrons uponinteraction 'it! p!otons ,asedon p!otoelectric effect.

# $lectrons are multiplied ,y a

series of dynodes# Proides current output

proportional

to lig!t intensity


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c. 8ey components + Doise# Jar current + Doise due to t!ermal generation-

increases 'it! temperature and !ig! oltage

# S!ot noise + proportional to t!e s2uare root of t!esignal


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L

ig!tIntensity

Maelengt!

LI*& SOU(C$

$fficiency

Maelengt!

"ODOC(O"6&O(

$fficiency

Maelengt!

P"&


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1,. Jistortions in e%citation and emission spectra# Lig!t intensity from lig!t source is a function of

'aelengt!

# "onoc!romator efficiency is a function of 'aelengt!

# &!e P"& does not !ae e2ual efficiency at all

'aelengt!s


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1c. Cali,ration# Correction of ariations in 'aelengt! of Renon lamp

and e%citation monoc!romatorDeed to do '!en measuring e%citation spectra or emissionspectra at multiple e%citation 'aelengt!s

# Correction of emission monoc!romator and P"&Deed to do '!en measuring emission spectra

I Fluorescence "easurements


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I. Fluorescence "easurements)a. $%citation 'aelengt! cali,ration

# $%citation spectra are distorted primarily ,y t!e

'aelengt! dependent intensity of t!e lig!t source

# Can use reference p!otodetector /cali,rated0 ne%t tosample compartment to measure fraction of e%citationlig!t

# &!e measured intensity of t!e reference c!annel isproportional to t!e intensity of t!e e%citing lig!t

Io

F

SampleCompartment

&o P"&

&o (eference P!otodiode


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I. Fluorescence "easurementsa. $mission 'aelengt! cali,ration

# Deed correction factors

# "easure 'aelengt! dependent output from acali,rated lig!t source

# Standard lamps of no'n and cali,ratedspectral outputs are aaila,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"&

SampleCompartment

Cali,ratedLamp


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,. $mission 'aelengt! cali,ration procedure# "easure intensity ersus 'aelengt! /I/00 of standard

lamp 'it! spectrofluorometer

# O,tain t!e spectral output data /L/00 proided for t!elamp

# Correction factor: S/0 L/0 I/0

# "ultiply emission spectrum 'it! correction factor


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c. $mission 'aelengt! cali,ration cure

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


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5. (outine e%perimental procedures# C!ec 'aelengt! cali,ration of e%citation

monoc!romator

# C!ec 'aelengt! cali,ration of emissionmonoc!romator

# C!ec 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

,ntensity

*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

,ntensity*

c.u.+

575 nm

!o'amine stan'ar'

scan



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a. Collection geometry in sample compartment# Front face + collection is at a )) degree angle relatie

to t!e incident ,eam- appropriate for an opticallya,sor,ing 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 transparentsample- less stray lig!t

Io F Io

F

Front Face (i !t 6n le


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d. Features of front face illumination# 6ppropriate for an optically a,sor,ing scattering

sample

# 6t !ig! optical densities9 lig!t is a,sor,ed near t!esurface of t!e cuette containing t!e a,sor,er- t!ereforefluorescence is detecta,le

# Fluorescence independent of concentration at !ig!optical densities


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. Blan scan# Blan is identical to sample e%cept it does not contain

fluorop!ore

# "easuring t!e fluorescence of t!ese samples allo's t!escattering /(ayleig! and (aman0 to ,e assessed

# In addition9 suc! samples can reeal t!e presence offluorescence impurities9 '!ic! can ,e su,tracted


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;. &ypical fluorescence emission spectrum at 37 nme%citation /t!e different components0

0

%00000

#000000

#%00000

2000000

2%00000

$000000

Fluorescence

Intensity

(a.u

.)

(aman

(ayleig! /e%c emm0

Fluorescence

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Introduction Fluorescence