time resolved fluorescence spectroscopy with a fast …
TRANSCRIPT
TIME RESOLVED FLUORESCENCE SPECTROSCOPY WITH A FAST ANALOG
TECHNIQUE: APPLICATIONS TO MICROSCOPIC SPECIMENS
by
MATTHIAS W. PLEIL, B.S.
A THESIS
IN
PHYSICS
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
Accepted
May, 1987
\ ^ ^ ^ ACKNOWLEDGEMENTS
I would like to thank the following individuals who have worked
closely wlth me in the lab and with whom I have had many fruitful
discussions: Greg Sullivan, Qiaries Landis, and Dr. Shubhra
Gangopadhyay.
I gratefuliy thank my mentor Dr. Waiter Borst for his guidance
ajid support; only through him was this work possible.
I also like to thank Drs. Roland Menzel ajid Thomas Gibson for
their advice and enlightening discussions.
I would like to thank my parents for their love and
encouragement.
Most importantly, I thank my wife, Oriajia, for her constant moral
support, love and encouragement. Without her I would not have
completed this work. I dedicate this thesis to her.
11
TABLE OF CDNTENTS
11 ACKNOWLEDGEMENTS
LIST OF TABLES iv
LIST OF FIGURES v
INTRODUCTION 1
aiAPTER
I. THEORY OF FLUORESCENCE 3
Li terature ci ted 18
II. APPARATUS 19
Li terature Ci ted 31
III. METHOD 32
Li terature Ci ted 55
IV. APPLICATION TO SCINTILLATORS 56
Li terature Ci ted 69
V. APPLICATION TO GEOLOGICAL SPECIMENS 71
Coal 71
Crude Oi i 75
Li terature Ci ted 86
VI. APPLICATION TO CRIMINALISTICS 87
Li terature Ci ted 93
CONCLUSION 94
COMPREIIENSIVE BIBLIOGRAPHY 97
111
LIST OF TABLES
2-1. Instrument Parameters and Fluorescence Signals 25
4-1. Scinti ilator Fluorescence Lifetimes 64
4-2. Summary of Effective Decay Times Determined by Others 65
6-1. Decay Times of Fingerprint Background Materials 89
IV
LIST OF FIGURES
1-1. Absorption of a photon by a molecule as governed by the Frajick—Condon principle 14
i-2. Possible paths of return to the ground state of a system in the exci ted s tate 15
1-3. The derivation of the single exponentiai fluorescence decay law applied to a collection of identical systems (molecules) 16
1-4. Fluorescence emission spectrum of Rhodamine 6G in ethanoi (0.15 g/L) by continuous wave mercury lamp excitation (365 nm line) 17
2-1. Schematic diagram of the apparatus 26
2-2. Internal side view of the Leitz MPV3 microscope 28
2-3. Digitized single photon response of the apparatus 29
2-4. Typical output from the photodiode trigger source 30
3-1. Graphical description of the convolution process 46
3-2. Total instrument response of the system 47
3-3. Totai system spectral response S(A) for a given optical configuration (20X Rolyn objective, K399 high pass filter, 0.5 mm exit slit. 0.3 mm measuring diaphragm) .. 48
3-4. Raw c.w. spectrum R (X) of a green-yellow resinite coal maceral 49
3-5. Spectrally corrected c.w. spectrum of the same green-yellow resinite maceral as in Figure 3-4 50
.3-6. Emitted fluorescence pulse of p-bis 2-(5-Phenyloxazolyl) benzene (or POPOP) in ethanol solvent fitted with a mono-exponential decay function 51
3-7. Self-deconvoluted instrument response assuming a mono-exponential decay 52
3-8. Time-resolved A-coefficient spectra and corresponding decay curves for seperate anthracene (T = 4.18 ns) and POPOP (T = 1.35 ns) solutions r>.3
V
3-9. Time-resoived fiuorescence spectra and decay times for an anthracene-POPOP mixture as a function of emission waveiength 55
4-1. Emitted fluorescence pulse from a Pilot-U scintillator fitted with a mono-exponential decay function 66
4-2. Dependence of fluorescence decay time on emission waveiength 67
4-3. Fluorescence spectra of the seven scintiilators obtained with continuous wave excitation 68
5-1. Fluorescence pulse emitted from an alginite maceral after laser exci tation 77
5-2. Example of time-resoived fiuorescence from an alginite coal maceral 78
5-3. Resolution of the composite fluorescence spectrum emitted from algini te macerais of New Albany shaie 79
5-4. A typical contiuous wave emission spectrum of an alginite coai maceral with the corresponding spectral parameters 80
5-5. Percent contribution to the total intensity averaged over the entire emission range as a function of sample maturi ty 81
5-6. Distribution of fluorescence decay times in coai macerals 82
5-7. Resolution of composite fluorescence spectra of two major maceral types found in the Kittaning coals 8.3
5-8. Fluorescence emission spectra and lifetimes from a Texas crude oi 1 84
5-9. Dependence of the three resolved fluorescence decay times (see also Figure .5-8) on API index for several crude oi 1 and condensate samples 85
6-1. Fluorescence spectra emitted from a cotton string fiber and from a f iber from a white sock 90
6-2. Fluorescence pulse emitted from a white sock fiber fitted with a bi-exponential fitting function 91
6-3. Fluorescence pulse emitted from a cotton string fiber fitted with a bi-exponential fitting function 92
VI
INTRODUCriON
A fast analog technique developed for the determination of
fluorescence emission parameters of microscopic geological particles
is applied to a variety of samples. Presented here are fluorescence
data from coal macerals. anaiysis of fluorescence emitted from crude
oils, applications to fiber analysis and piastic scintillators.
Characteristic decay times in the nanosecond and subnanosecond range
are given along with time-resolved emission spectra.
The new fast analog technique enabies us to resolve individual
fluorescing components from a mixture of one, two, or three
non-interacting fluorophores by differentiating between the individual
lifetimes. The microscopic samples are excited with a near
uitraviolet pulsed nitrogen-pumped tunable dye laser. The emitted
fluorescence is detected at specific wavelengths with a micro-channel
piate photomultiplier tube and fast waveform digitizer. A number of
pulses (64-2048) are signal averaged and passed on to a computer for
data analysis. Using iterative reconvolution techniques and least
squares fitting, the instrument response is removed and the
fluorescence emitted from a variety of sampies is modeled with a sum
of exponential terms, each term corresponding to an individual
fluorophore. Complex systcms are characterized by rcsoiving the
individual fluorescence spectra and lifetimes.
This technique has been under deveiopment since the beginning of
1983 by W. Borst ejt aT. The author joined the project as an
undergraduate in November of the same year. Much of the author's time
in research has been spent on learning the operation of the equipment,
developing software for data acquisition, anaiysis, and calibration
routines, checking and rechecking the quality of the procedures
developed and maintaining ail of the equipment in peak operating
condition. From the outset, the physics group has been working in
cooperation with the geology departments of Southern lilinios
University (1983-1986) and Texas Tech University (1986-present). As a
result of this cooperation, the system was designed for ease of use,
requiring as little as possible of the operator in terms of technical
skills, allowing him to concentrate on the samples under investigation
and enabling him to acquire large amounts of data, which is necessary
in geological analysis. The system is routinely used by Charles
Landis, the resident geologist, who has provided the group with a
weaith of geological data and who has directed the geological
research. The technique has been tested under diverse sample
conditions and its limitations determined. It has also been appiied
to other specimens more directly related to physics including plastic
and acrylic scintillators, and various fibers.
CHAPTER I
THEORY OF FLUORESCENCE
Luminescence properties are extensively used for characterization
of materials. The term 'luminescence' is broad and covers all
phenomena related to the emission of light from materiais which have
been excited by the absorption of radiation or by other means.
Luminescence is made up of two catagories: phosphorescence and
fluorescence. Fluorescence is a fast phenomenon which stops
-9 practicaily at once when the excitation source is removed (10
seconds) and it is the result of a transition between two electronic
states having the same spin multiplicity. Fluorescence is commonly
observed in gases, solutions, and solids. Phosphorescence is a slow
phenomenon and is the result of a transition between two states of
different spin multipiicity; it is primariiy observed in solids where
the re-emission of radiation may continue for hours [1].
Energy must be absorbed before luminescence can occur. Whether
or not light of a given frequency can be absorbed by a moiecular
system is governed by the Franck-Condon principie. Figure 1-1 is a
plot of the potentiai energy as a function of internuclear separation
of a diatomic system. The lower curve is for the ground state
configuration (S^). The upper curve is for the first excited singlet
state (S.). The closely spaced horizontal lines within both potential
curves represent the various vibrational modes the system may be in
for a given electronic state. The Franck-Condon principle basically
states, that, since the absorption process takes very little time (on
-15 the order of 10 seconds), the absorption from the ground state to
the first excited state must be represented by a vertical line. In
other words, the system does not change its internuclear configuration
during the absorption process (the internuciear seperation, R, is
constant). At room temperature, the occupation of vibrationai states
is governed by the Boitzmann distribution:
^ ^ e"^^^'^ (1-1)
or equivaiently:
^= -(E2-E,)/kT (J.2)
where ^ (=N /N ) is the ratio of the population of level 2 cind level 1
and AE (=E„-E.) is the energy difference of the two vibrational states
in question. T and k in the above equations represent the temperature
and Boitzmann's constant, respectively. If one lets T=300 K, and
AE=i500 cm~ (approximately the spacing of two successive vibrational
energy levels), then ã = O.Oi. Hence, the molecule will be in its
lowest vibrational state at room temperature [2]. Since the molecule
is most probabiy in its lowest vibrational levei in the ground state
prior to absorption (v=0, S ), the internuclear separation will have
its equilibrium value R . Note, for higher vibrational levels (v>l).
the probability is that the internuclear separation will be close to
the ailowed extremes of the potential curve (this is analogous to the
harmonic osciilator where it is found from the wavefunction that the
probability distribution for the particle is highest close to the
cxtremes of its motion for v>l). Quantum mechanical iy, the lare;csL
.3
overlap between the two wavefunctions involved in the absorption
occurs for the strongest transition (in Figure 1-1, the most probable
transition is S„, v=0 —* S^ , v=4).
Once the system has gone through the absorption process and is in
the excited state, it may go through vibrational relaxation or it may
emit a photon from the vibrational level to which it was initially
excited (through spontaneous or stimulated emission). Wliich of these
processes is dominant depends on the environment of the system. In
the gas phase, a given molecule is essentiaily isolated, hence, the
only way for it to iose vibrational energy is to either emit an
infrared photon (ending up in the lowest vibrational level of the
first excited state) or it can make the transition directly to the
ground state. The direct transition to the ground state is the most
probable, hence, in rarified gas phase studies, the vibrationai levels
of the excited state are easily discernible in the emission spectra
obtained by broadband excitation. The work presented here consists of
liquid and solid phase studies. A collection of fluorophores
(fluorescing molecules) in liquid or solid phase lose all excess
vibrational energy in the first excited singlet state prior to the
emission of a photon. This energy transfer is accomplished by thermai
relaxation through interactions wilh other molecules and is very
-13 -11 rapid. 10 ' to 10 seconds [3]. Therefore. absorption. inciuding
vibrational relaxation, can be considered instantaneous with respect
-9 -3 lo the emission process (~10 - 10 scconds).
Shortly after absorption the systcm is in the lowest vibrational
mode of the first excited singlet slate. Tlie systcm can return to thc
ground state via radiative or nonradiative paths (see Figure 1-2). If
the transition is direct from the first excited singlet state to the
singiet ground state and involves emission of a photon, the phenomenon
is termed fluorescence. If there is inter-system crossing. where the
first exciled singlet state makes a transition to a triplet state of
iower energy (through spin-orbit interactions via vibrational coupling
between the two states), and then an emission of a photon occurs as a
result of the T. -» S„ transition, the phenomenon is termed
phosphorescence. Of course the system may also return to the ground
state (in general, any of a number of lower states) through numerous
non-radiative paths which invlove energy dissipation through phonon
interaction with the surrounding media (consequently, the system
environment heats up); this is called inLernal conversion in the case
of no change in spin muitiplicity (As = 0) and inter-system crossing
in the case of a change in spin muitiplicity (As = 1). Energy may
aiso be transferred to other molecular systems by dipoie-dipole
interaction and they in turn may or may not radiate.
The radiative transition from thc first excited singlet state to
the ground state in an atom is governed by the quantum mechanics of
spontaneous and stimulated emission. Spontaneous emission occurs
without any externai perturbation (hence the term spontaneous).
Stimulated emission is the rcsult of an external electric field
perturbing the system thereby inducing the transition to a lower
electronic state resulting in the emission of a photon. A proper
quantum mechanical treatment of the absorption and emission proccesses
would involve the use of quantum elcctrodynamics (the quantization of
the electric field). However, one may use Einstein's detailed
balancing approach coupled with the electric dipoie approximation to
get an accurate description of the emission processes involved [4. 5].
The developement of detailed baiancing is applied below to the
simpiified case of a two state electronic system; there is no
degeneracy nor is there any interaction by collisions with other
systems.
Detailed balancing makes use of the fact that the induced
absorption rate is
R(^.^^^ =B...p A(o..) (1-3) ij ij rad'' ij^
where B. . is the Einstein "B" coefficient and p ,(w. .) is the energy ij rad^ ij'
density of the radiation:
"radt^ij^ = (l^l^/^TT) . (1-4)
ã is the magnitude of the electric fieid. The excitation light has to
be of energy hv = AE where AE is the energy difference between the two
electronic states i and j for absorption to be allowed. The frequency
of excitation must therefore be w. .(= 2TTV) . The Einstein B
coefficient can be approximated by
^^ 3 \i •-'
using the first order electric dipole approximation where
e = the charge of an eiectron
h = Planck's constant/2F
új = the wavefunction of the i and j states i.J
and
r = the average position vcctor of thc electron and is in the
direction of the electric dipole moment (p = e*£. where p is the
dipoie moment) [4] <//. |i:p .> is the matrix element and is a measurc
of the degree of coupling between the two states.
The spontaneous rate for electronic transitions. Ni/.> to !>/'.> J ' 1
states, can be written as
j^(spont) ^ ^ >j,
Ji Ji ^ J i' (1-6)
where A.. is the Einstein "A" coefficient and E., E. are the energies Ji J 1
of the j and i states, respectively.
The number of molecuies making the transition from the lower to
the higher energy state is
dN. . ^-J.^N.-R?^.^^) =N.B .-. . p ,(w. .)
1 1j rad 1j (1-7)
dt 1 ij
and the number of transition being made from the higher to the lower
state is:
dN. . - r ^ = N . dt j
j (stim) ^ j (spont)
ji Ji (1-8)
where R Ji
(stim) . , ^ r . 1 . 1 • • n(stl" ) ^ '^ is the rate for stimulated emission. K. .
is Ji
related to the energy density p i w ) by'-
„(stim) „ , .. R.. ^ = B.. p j(w ..) ji ji ' rad^ ji'
where
"^ 3 h^ '
and is cailed the Einstein B coefficient for stimulated emission
(1-9)
(1-10)
Hence,
dN. . J-^i
d t = N.
J B..P ,((.)..) + A.. ji' rad' ji' ji
(1-11)
In equilibrium. the time rate of change of the number of molecules in
the upper and lower states must be equal. Therefore.
dN. . dN . . (1-12)
dt
or
N. J B. .p ,(w.. ) + A.. j r rad^ ji'' ji
= N.B. .p ,((j. .) 1 1j rad 11'
(1-13)
Taking the ratio of N . to N. J 1
N. _i _ N. " 1
B. .p ,(o). .) ij^rad^ ij^
B . .p ,(GJ . . ) + A .. ji'^rad^ ji'' ji
(1-14)
and equating it to Equation (1-2) (the molecular system is in thermai
equilibrium with its environment), one establishes the result:
B. .p ,(w. .) ij'^rad^ ij^
B. .p ,(w.. ) + A.. ji' rad^ ji^ ji
We have w. . = w.., and ij Ji
p .(w. .) = p ,(w ..) rad 1 j rad j i'
Solving for p ,(w..), rad j 1 •'
= e -(E.-E.)/kT ^"^jiXkT
j 1' = e ^ (1-15)
(1-16)
P j(í^--) = rad j1' JLl
B. . e^^'j^'^^'^ - B
(1-17)
ij Ji
and noticing that the energy density follows Planck's distribution
law
~ 3 h(i).
Prad<-'ji) = Vî •
the relation:
A . . âl
h(0 . . Xrr
B. . e J ' / ' '^ - B
TT C hcJ . . / , .T-
e J ^ ^ ^ ^ - 1
(1 -18)
1 h o j . . J ^ .
2 3 --ir c h ( i ) . . / , .T-
e J^-^^^ - 1 ij Ji
is obtained. Here, c is the speed of light in vacuum.
(1-19)
Thc only way
that Equation 1-19 can be true, is if Lhc following relations arc met:
B.. = B. . Ji iJ
(1-20)
and
10
A .. h(ú ..
B. . 2 3 (1-21)
IJ TT C
or equivalentiy,
'"' 3 h(i)..
ji ij 23 vi - - ; TT C
Note. the detailed baiancing results given above apply to the gross
behavior of a molecular system. Originally, this approach was applied
to electronic transitions as would occur in rarefied gas studies of
atomic systems. If one denotes the degeneracy of the electronic
states |v//> and \yp .> by g. and g., respectively, then Equation (1-20) becomes:
giB.. =g.B.. (1-23)
while Equation (1-22) remains the same with B. . replaced by g.B. . [.51. ij J ij ^ -
In time resoived studies, one looks at the fluorescence of the
specimen after the excitation source has been turned off. In most
cases p .((j. .) is negligible and essentially nonexistent (except
wi thin a laser cavity where p ,((J. .) can be appreciabie) . This
results in the depopulation of the excited state being governed only
by the spontaneous emission rate A... The radiative lifetime of the
system is just the inverse of the spontaneous emission ratc:
T " , = A.. . (1-24) rad j 1
The fluorescence lifetime observed from a specimen by measuring the
photons emitted in time includes contributions from nonradiative as
well as radiative rates.
The average amount of time a molecular system is in the excited
state can reveal much about its environment. By exciting N sysLems
into Ihe excited state and observing the emission of photons with
11
time, one may determine parameters such as Lhe decay time of the
system in the excited state, which includes non-radiative as well as
radiative decay rates. For most systems the probabiiity of emission
is directly proportionai to the number in the excited state. hence,
the emission follows a single exponential decay law. The number of
photons emitted in the time interval from t to t+dt is dN where dN is
proportionai to the number of systems (molecules) in the excited state
N:
dN = -R-N-dt . (1-25)
R is the rate constant governing the efficiency of transition from the
first excited singlet to the ground state and the right side of the
equation is negative indicating a reduction in the number of systems
in the excited state with time. R takes into account radiative and
nonradiative energy transfer rates including intersystem crossing.
Figure 1-3 shows the derivation of the single exponential decay law.
Since the intensity emitted from the collection of systems is directly
proportional to the number of photons emitted at any specific time,
one may write for a single fluorophore:
I(t,A) = I^(A)-exp(-t/T) , (1-26)
where I(t,Á) is the intensity of the flourophore at time t and
wavelength A, I (A) is the intensity at t=0 for the given A and T is
again the fiuorescence or observed lifetime. In the absence of
competing processes, where all non-radiative rates are suppressed. the
observed decay time T is just the intrinsic or radiative lifetime of
the excited state ~^^..^^-
12
The extension of the above derivation to a number of non-
interacting fluorophores is straight forward. For N. non-interacting
fluorophores, the intensity as a function of time at a given
wave1eng th i s
N
I(t.A) = ^ A.(A) • exp(-t/T.) . (1-27)
i = l
Here A.(A) is the pre-exponential coefficient and it is equivalent to
I (A) in Equation (1-26) for the individual fluorophores.
Most organic specimens are made up of a conglomerate of N
fluorophores which emit a total signal I(t,A). If one can determine
the individuai A.'s and T.'S which make up the total I(t,A) over a
wavelength range, then one may separate the conglomerate spectrum into
its components, hence, the individual constituents may be identified.
The absorption and emission spectra for most organic molecules in
the liquid or soiid phases are in generai quite broad at room
temperature. These spectra show very little structure. The lack of
weil defined peaks, which would correspond to transitions between
vibrational levels, is a result of the large number of vibrationai
levels and rotational sublevels. The rotational levels are also
extremely numerous at room temperature. hence. the resulting spectra
are effectiveiy continuous.
Figure 1-4 shows a typical emission spectrum of a pure substance
(Rhodamine 6G at 0.15 g/L concentration in ethanol). The peal< at 566
nm represcnts the emission from the lowest vibrationai level of the
first excited singlet state (this experiment was done at room
temperature) to an upper vibrational level of the ground state. This
13
most probable transition of emission corresponds to an energy
difference of .576 nm or AE = 17361 cm . Thc emission spectrum is
red-shifted relative to the absorption spectrum due to Stokes' shift.
The primary reasons for Stokes' shift is 1) there is some cnergy loss
due to vibrationai relaxation when the system is in the first excited
singlet state, 2) the first excited state of most molecules decay into
an upper vibrational level of the ground state due to the
Franck-Condon principle (see Figures 1-1 and 1-2) and 3) the excited
molecule becomes more polar resulting in a reorientation of the
surrounding solvent molecules which in turn lowers the energy of the
system (the energy gap between the two states decreases) [2]. The
vibrational levels of S„ and S.. are usually very similiar and the
reciprocal transitions have proportional probabilities (i.e., if the
S, , v=0 -* S v=4 absorption transition has the highest probability.
so does the S^. v=0 -» S„, v=4 emission transition, see Figure 1-1).
As a result, the absorption and emission spectra are oftentimes mirror
images of each other. This is indeed the case of Rhodamine 60 in
dilute ethanol soiution [6].
The work presented here depicts a fast analog technique used in
determining the pre-exponential coefficients and corresponding decay
times of fluorescence emitted by complex organic systems made up of
one, two or three non-interacting fluorophores. From these
parameters. the component spectra may be resolved allowing one Lo
"fingerprint" Liie organics cind in some cases even determine Lhe
chemical composilion of these unknowns.
14
P o t e n t i a 1
E n e r g y
Thc Franck-Condon l ' r i n c i p i e
F i r s t Exci tcd
S i n g l e t S t a t e
Ground S t a t e
= G
= 6 5
1 0
i
0
Internuclear ScparaLion
Figure 1-1. Absorption of a photon by a moleculc as govcrned by the Franck-Condon principle. The graph reprcsents the potcntial energy curves (not to scaie) as a function of internuclear separation for the first excitcd singlet and ground sLatcs of the bond rcsponsible for fiuorescence. The horizontal lines represent the vibrational modcs (rotational modes are not given for clarity). The absorj^tion and emission takes piace over negligible timc, hencc, the sysLem docs not changc its internuclear scparation. As a resuit, vertical lines are drciwn to represent the change of eiectronic state. In this dcpiction, the molecule is at room temperature.
15
Possiblc Excitcd —* Ground SLate Energy Transfer Path;
«1 -
Vibrationai Rclaxation
Fluorescence Emission
or Internai
Conversion
o
Inter-system Crossing
- T,
Phosphorescence
or Internai
Conversion
Process
Exci tation
Rela.xation (Internal conversion)
Inter-system crossing
Phospiiorescencc H
l'luorescencc
Time (seconds)
=;io-'-^ ;.io-^2
:.10-'2 -3 4 -10 - 10
-9 - 10
H Timcs include contribution from non-radiative transitions
i- igurc 1-2. i'ossiblc patlis of rcturn to thc ground sLate of a systcm in tlie excited staLe. Oncc cxciLed, tiie systcm gocs tiuougli vibrational relaxation jîrior to inter-sysLem crussing or dirccL rcLurn Lo tiie ground state. Return to ' iie ground state may or may not include emission of radiation. 'l'imes associatcd witií tlíc various processcs are also givcn.
16
Ratc of Decay of an Initially Excitcd StaLe
dN(t) _ dt
l = -(k + k )'N(t)
i^adiaLivc Non-I^adiaLive Dccay RaLcs
NumÍDer of Molecules i n the c x c i t e d s t a t c
i^ewr i t ing :
M>,( = - ( k + k ) d t N ( t ) ^ r n'
In l . cg ra t i ng :
N(L) = N -e o
-L/T
1 r = (I< + k ) and N is Llie numbcr of cxcitcd molccuics at t=0, ^ r n^ o
I'igure 1-3. 'I lie derivation of tiie single exponential fluorescence dccay law applied to a collcction of idcntical systcms (moiccules). T is the decay time or lifetime of the molecule in Liie first excited singlet sLate in Lhe presence of competing. non-radiaLive processes. T is Lhe Lime iL Lakes for Lhe number of excitcd molccules Lo decrease Lo 1/c of Liic iniLial numbcr of cxciLcd sysLcms, and r is commonly referred Lo as Liie observed lifetime.
17
>-»—
> h-<
I
LU CC
1.0
0.8 .
0.6 .
0.4
0.2 •
0.0 400 500 600 700
WAVELENGTH (nm)
800
I-'igure 1-4. F luo re scencc cmiss ion specLrum of i^hodamine GC in e t i ianol ( 0 . 1 5 g/L) ijy con t inuous wave mercury lamp c x c i t a L i o n (.3G.5 nm l i n c ) . Tlie specl rum iias Ijcen smooLlicd and normalizcd Lo uni t I ie ight ; i ts pcak o c c u r s at .566 nm.
18
Literature Cited
1. Hirschlaff, E. Fluorescence and Phosphorcsccnce, Chemical Publishing Co. Inc., New York, 1939.
2. Lakowicz, Joeseph R. Principles of Fluorescence Spectroscopy. Plenum Press. New York, 1983.
3. Hercules, David M. Fluorescence and Phosphorescence Analysis. Interscience Publishers, New York. 1966.
4. Weider, Sol The Foundations of Quantum Theory, Academic Press, New York. 1973.
.5. Bransden, B.H. and Joachain, C.J. Physics of Atoms and Moiecules, Longman, London, 1983.
6. Berlman, Isadore. B. Handbook of Fluorescence Spectra of Aromatic Molecules, Academic Press, New York, 1971.
aiAPTER II
APPARATUS
The laser fluorescence microscopy system is represented
schematicaliy in Figure 2-1. Further specifications are given in
Table 1-1. A pulsed laser and three sources of continuous wave (c.w.)
illumination are interfaced with a Leitz MPV3 microscope system. The
nitrogen pumped dye laser provides intense near-ultraviolet light
pulses. The laser is coupled to the microscope via a liquid light
guide. The modular construction of the microscope system allows
various optical components to be readily interchanged. The emitted
fluorescence is detected by a fast two-stage microcliannei plate (MCP)
piiotomul tiplier tube. The output of tlie MCP is directed either to a
picoammeter when acquiring conventionai c.w. spectra or to a fast
waveform digitizer when acquiring time-resolved spectra. The
digitizer sums a number of MCP output pulses and passes the average to
a desktop computer where data reduction and anaiysis take place.
Four light sources are linked to the microscope. The three c.w.
sources are part of the Leitz MPV3 system. The tungsten lamp
(Philiips 100 W #7023), mounted directly onto the right side of the
microscope body, is used primarily for spectral calibration. However.
it is also used as a white liglit source for viewing the samples
because i ts output is primarily in tlie visible part of tlie spectrum.
Thc 75 W high pressure xcnon lamp is a broadband source cmittini:!:
continuously from the ultraviolet tlirougii the visible parts of tlie
19
20
spectrum. The intensity of the xenon lamp at any particuiar
wavelength is much lower than the 365 nm line of the high pressure.
100 W mercury arc lamp (IIBO-lOOW/2). The ,365 nm line is selected by
the input monochromator and it is the primary c.w. fluorescence
excitation source. This line was chosen for its relatively high
intensity and its wavelength is in the near-ultravioiet (u.v.) making
it compatible with the glass optics on the excitation side. (Glass
absorbs in the u.v.) The iaser excitation line was chosen close to
this wavelength for comparison purposes. The output of the input
monochromator is directed through a collimator and enters the left
side of the microscope. The collimator contains a collimating lens
and a turret, which has three settings, two of wliich transfer filters
into the light path (380-800 nm or 200-390 nm band pass), and the
remaining setting is just an open diaphragm. The monochromator and
collimator are bypassed when a higher intensity is desired by mounting
the lamp directly to the microscope housing.
The EG&G 2100 nitrogen-pumped tunable dye laser may utilize any
of a number of dyes ailowing a wide range of pulsed excitation
wavelengths to be used. The nitrogen laser pumps the dye laser with
1.2 ns FWHM, 337 nm, 0.6 mj pulses at a repetition rate of 1-100 pps.
The dye is in a standard fluorescence quartz cuvette situated within
the laser cavity. This cavity uses a grating set at grazing incidencc
and a totaiiy reflecting mirror. which is coupled to a stepping moLor.
This combination resulLs in a tuning range of .360-800 nm. Utilizing
BPBD dye. the laser provides ncar u.v. pulses at 373 nm and is tiie
source of fluorescence excitation for timc-resolved spectral analysis.
21
The full width at half maximum (FWHM) of an individual dye laser pulse
was determined to be about 500 ps by a standard time-correlated single
photon counting technique presently under developement. The energy
per pulse was found to be greater than 10 pj using standard
calorimetric measurements and a Scientech 362 calorimeter. The iaser
generailly operates at a repetition rate of 10 Hz.
The laser pulses are directed via a iiquid iight guide to the
rear of the microscope. Figure 2-2 gives a cut-away side view of the
microscope interior. The laser pulse enters the microscope (left side
of Figure 2-2) and bypasses mirror M . which is removed from the path
for laser excitation. (M. is in place when exciting the sample with
c.w. sources. M^ allows one to chose either the right or left ports of
the microscope housing. Proper adjustment of M^ and M^ also allows
one to chose between transmitted or reflected illumination of the
sampie). After passing through the diaphragm D^, which is situated
between the two excitation lenses L. and L^, the laser pulses are
directed through the illumination diaphragm ID, and fieid diaphragm
FD. The laser beam is out of focus at the illumination diaphragm,
hence, by varying its diameter, one effectively controls the intensity
of excitation. The excitation beam is focused at the field diaphragm
ailowing one to continuously adjust the size of the illumination spot
on the sample (when the objective is focused). After passing through
tiie field diapliragm, tlie excitation liglit is reflectcd to the sample
by eitlier a diciiroic mirror or a neutral density beam splilter. The
dichroic mirror reflccts all (>90%) light below 400 nm and ailows
light above 400 nm to pass through. Tlie nuetral density beam spliLter
22
reflects 50% of tlie incident light regardless of its waveiength
(.300-800 nm). The dichroic mirror is used when coilecting
fiuorescence data while the l eam splitter is used when determining the
instrument response, which includes the laser pulse characteristics.
These mirrors are each part of a "cube" and there are four cubes
making up the "Ploemopak," which is a turret of cubes allowing for
easy exchange of optical elements. The Ploemopak is unique in the
MPV3 construction, for it allows the user to interchange optics
readily by rotating a knurled knob, which effectiveiy places one of
four opticai cubes into position. Tlie laser pulses are reflected down
through the objective and then onto the sample. The light excites the
sample fluorescence, which passes through the objective and dichroic
mirror, (the emitted fluorescence is generally above 400 nm) or
through the beam splitter. A K.399 high pass filter (Oriel) is used
when acquiring fluorescence above 400 nm (F. in Figure 2-2) to cut out
any leakage from the Hg lamp (or laser), which shows up as a second
order contribution in the range of 725-745 nm and is appreciable for
low intensity samples. The light is either directed to the oculars,
top port (used in taking photographs of the sample) or to the exit
monochromator. Before reaching the exit monochromator, tiie
fluorescence must pass tlirougli the measuring diapiiragm whicli allows
the user to select the area on the specimen he wishes to measure.
Tiiis diaphragm can be chosen to be either circular with incrcmental
diameters from .0'/" to 5 mm or in may be rectangular with continuously
variable widLÍis and lengths in the same rangc. Tlie image of the
measuring diaphragm can be seen ly the user through the oculars if
23
desired. The measuring diaphragm may also be rotated to fit
irregularly shaped specimens; this is done by actually rotating the
image under investigation using the prism arrangement situated in
front of the measuring diaphragm. Hence, one can be very specific
about the measured sample area; samples down to a few microns and up
to hundreds of microns in size have been easily selected and their
fiuorescence measured. The wavelength to be detected is selected by
the exit monochromator. This grating monochromator is in Littrow
arrangement and has 600 lines/mm resulting in a dispersion of 6.6
nm/mm exit slit width [1]. Interchangeble slit widths of 1.0, 0.5,
0.3, and 0.15 mm results in a resolution range of approximately 7 to 1
nm, respectively. The principle wavelength is scanned lineariy and
continuously either manually or by two computer controlled motors.
Tlie wavelength at which the monochromator is set is recorded by the
computer with an analog to digital converter from a potentiometric
output.
The Hamamatsu R1564U-01 two stage, proximity focused microchannel
plate (MCP) photomultipiier tube detects the wavelength-selected
fluorescence. This state-of-the-art MCP has 12 pm in diameter pores
resulting in a transit time jitter of approximately 90 ps [2]. Figure
2-3 represents a typical singie photon (or delta function) response of
the MCP convoluted with tlie digitizer response when a potential
difference of 3.0 kV is applied.
Tlie output from the MCP is directed either to a picoammeter when
acquiring continuous wave spectra. or to a Tektronix 7912AD fast
waveform digitizer when acquiring fluorescencc decay data. Fhe
24
picoammeter converts the MCP output current (-- nA) to a 0-10 V signal
appropriate for the analog to digital (A/D) converter of the computer.
Tlie fast waveform digitizer consists of tiie main control uni t (the
7912AD) and two plug-in units, the 7129 amplifier and the 7890P
programmable time base. The frequency response of the input amplifier
is about 1 GHz, and the maximum digitization rate is 100 GHz (10
ps/channei). We generally use the 40 ps/channel rate. The vertical
scale is divided into 512 channels (and so is the time scale). A
photodiode monitors the laser pulses and triggers the digitizer. A
typicai photodiode output pulse (convoluted with the digitizer
response) is shown in Figure 2-4. The individual phomultiplier pulses
are summed by the digitizer. The number of pulses ranges from 1 to
64. For acquisitions over 64 pulses, the digitizer sums in blocks of
64, sending the average of each block to the Tektronix 40.52A computer.
The computer system includes a Gemini-10 printer (Epson), Hewlet
Packard 7475A 6-pen plotter, Tektronix 4631 hard copy unit, three
Tektronix 8 inch floppy disk drives. a 40.50E01 ROM expEuider and a
built in memory expansion unit. which is used for data storage and
programming.
The entire system has been designed to allow easy data
acquisition. storage and analysis. With the above mentioned
components we can routinely acquire c.w. fluorescence and
time-resolved specLra of microscopic specimens. The relative ease of
use faciIiLates interaction with other. lcss technical ii,roups and
allows greater precision in repeatabi1ily of experiments.
25
Table 2-1. Instrument Parameters and Fluorescence S]
Size of sample Diameter of analyzed area Tuning range of emission monochromator Range used Monochromator bandwidth
Continuous Fluorescence Excitation
Tuning range of excitation monochromator Typical excitation wavelength Monochromator bandwidth
Fiuorescence Excitation by Pulsed Laser
Dye laser pulse duration (FWHM) Puise energy (BPBD dye, .37.3nm, lOHz) Peak power Laser bandwidth Dye iaser tuning range
Excitation wavelength used Pulse repetition rate Photons per pulse (BPBD dye) Photons reaching sampie Photons onto measured region C^ 10 pm)
Typical fluorescence yield Photons reaching MCP Typicai number of photoelectrons per pulse Instrument response risetime FWHM of instrument response Single pulse digitization rate Number of pulses signal averaged
10 5
220 380
220
.370
1
Lgnals
- 750 pm - 2.50 pm - 800 nm - 750 nm 1 - 7 nm
- 800 nm 365 nm
1 - 7 nm
< 600 ps > 10 nJ > 10 kW 0.04 nm - 780 nm
.373 nm - 100 Hz > 10''' > 10'^ - io'°
0.001 - 0.3 lO '• - l O ^
10 - lO''
<
10 -
< 700 ps . 1.25 ns - 100 GHz 64 - 2048
26
HARU
COPY UNir COMPUTER
IN/OUT
DEVICCS
XENON ARC LAMP
MERCURY ARC LAMP
PLOTTER
PRINTER
WAVEFORM DIGITIZER
MONOCHROMAÎOR
TRIGGER
MCP
MONOCHROMATOR
LEITZ MPV 3
MICROSCOPE
TUNGSTEN
LAMP
TUNEAULE
DYE LASER
NITROGEN
PUMP LASER
Figure 2-1. Schematic diagram of the apparatus. Fluorescence puises from the sample surface are excited by pulsed laser. spectrally analyzed, and acquired with the fast waveform digitizer for subsequent time-domain analysis by computer. For continuous wave excitation, mercury and xenon lamps are used and a picoammeter (not shown) is connected to the MCP photomuitiplier and computer.
27
(D
m 3; X <T> M. P n- W
C W '1 »—' M .
!-• 3 f^ Oq
CL M -
P "a D' n P oq 3
n: • - < •
ap D '
• 0 P U) U)
• - • í M .
• —
r t (\> -1
< • — ^ 50% at 399
nm. ~96%
above 401
nm
0 cr C_i.
a> 0 r-r (-!• < ft>
' o ' M .
0 Í T 1 0 (-•• 0
3 >-•• 1 T 0 - 1
0 -J
neutral density beam
splitter
*r) M -
n> M '
a a M -
t3 ir - 1 P
09 3
MH ^ ^ ^ .
c 3 M ^
3 fi3 r-t M .
0 D
a M .
&3 'a =r-ragm
r a> D W fD W
S M .
•1 '1 0 1 (/)
(A fD (—' a> o r t n> a
a
w (fl a) a
o fa
a :r a 0) M.
o 0) ar X n M. O rt M.
o 3 O 3 3 M. O n O -î p f O -1 -1
•-n -1 O 3
a> o - j
a
(/)
o - 1 o o nr P
a>
•d P <-r 0)
X}
o (-r O 3 C
X)
a) - 1
tr a fD M. -1 -1 0) a>
o r-r <-r D ' a> a> a í a> (a M. < <-r a> rr H - a> fD -1
09 <-r <-r O :r
<-r rt :r O 0) cr o fl> o
c a M-0) P3 <-T T a> (fl o rr » - • 1
a> o a -j
c cr a>
(/) a> M.
oq
•a 3 O M
f-r -J O
fO - r t
(fl 3 : a> O to
X P P a> rr P 3
a o 3: p • r r p
rr a c/) a> P 3 3 • - 0»
*0 ••'• -J •—09 O 0) :r c
r r -J '-'1 »<
C r r M-O C P -! D 3 (D 09 t3 (/)(/)(/! O <-r fD fD fD 3 3 P O <-r fD •— fD
P -! r r 3 (fl - 'O P w r-r < nr (D fD -J •— P O (/) <-r C
0» 09 C -I r
Xí (fl <-r
r r r r pr'
:r zr (D -J -J o o •— C C 0)
09 09 -•^ = r D- r r <-r <-r " 0 =r D- o (D fD -J
<-r O -1 cr M. p
CM. 09 r r 0) n r O <-r
rr p <-r :r D-(D (/) -1
r r O cr -J C 0) P 09 p M. izr 3 09
:r r r M. r r l ^ (fl fD
•o a p 1 M. <-r 0)
< ir p fD "1 T '—» r r 3 '--s fD M. K— a 1 0) O O <-r 3 -J rr (/) 0 3 : —
a =r M^ fD U)
0)
(/) -J p 0) a 3 3 •o o — < • a 3
^ 0 9
P
M. M.
09 09 :r c <-r -J
(\) Xi p ro <-r I :=r to
(/) >—i
M. ID 3 r t
a a> M. -1 o 3 P P <-r ^-a> a (fl
M cr a '^ a> r r < 3 - — 0) a> a o o
fD r t a 3-
O r r M. r -O O O 3 fD 3 'i-) fD M.
M. i r 3 0) c o a C M. (n O
3"
r t N 3" a> H 3 :
3 - -T3
3 a> <
p < M. (D O
^- 3
C 3 M' 3 P
O 3
-J -O - J
« 3" 0) - j (D
(fl fD O T O
TI 0)
< a>
P 3 (/) >-••
o -J o
M- U) M. n
09 O P 3"a 3 <-r a> a ••n 3 O <-r H- fD •—' -J
O (A
(/)
H 3-0)
28
oc ^ '^ O uo a: <
29
=3 Q .
•— ' - '
Z) > O E Q_ ^ \J
-
4.99
3.90
2.81
1.71
0.62 -
- 0 . 4 7 '
SINGLE PHOTON INSTRUMENT RESPONSE
RISE TIME 0.52 ns
FALL TIME 1.00 ns
1/2 WIDTH 0.86 ns
y V - ^ jirui ^r^^^
12 r
16 20
NANOSECONDS
Figure 2-3. Digitized single photon response of the apparatus . This pulse represents the convolution of a delta function signal (photon) with the response of the detector. which is made up of the 2-stage microchannei plate photomultiplier tube. RG-223, 50 Q coaxial cable and fast waveform digitizer.
30
124
99
> E
Z) CL.
74
O 49 UJ (21 O 5 24
TRIGGER SIGNAL vs. TIME
RISE TIME: 1.55ns
FALL TIME: 6.23ns
TIME (ns)
Figure 2-4. Typical output from the photodiode trigger source. The diode is situated close to the dye laser head and detects the back reflected laser signal. The photodiode output can be changed ly varying its position in the reflected beam.
31
Literature Cited
1. E. Leitz Inc. Product Literature Detailed List of Microscope Equipment and Accessories, Part No. 610-126 p. 5/5 1983.
2. Hamamatsu Photonics K.K. Proximity MCP PMT Data Sheet.
CHAPTER III
METHOD
The acquisition and reduction of the pulsed fluorescence data
requires much care. The fluorescence decay received by the computer.
M(t), is not the actuai fluorescence signal emitted by the specimen
F(t). M(t) is the convoiution of F(t) with the instrument response
function, I(t).
The individual fluorescence pulses emitted from the specimen.
F'(t). are actually a convolution of the laser pulse profile, L(t),
with the actual fluorescence decay of the specimen, F(t). The
convolution of the laser pulse with the fluorescence decay is
represented in Figure .3-1. The mathematical analog to the
laser/fluorescence folding ( or "faltung") can be represented by the
convolution integral:
t F'(t) 3. / L(T).F(t-T) dT
— 00
= L(t)^F(t) (3-i)
The MCP itseif will foid its temporal response, Mcp(t), into the
signai it detects, its final output being S(t):
t S(t) = / Mcp(T)-F'(t-T) dT . (-3-2)
—00
The MCP signai is transfered iy a RG-223. .50 Q coaxiai cable. which
distorts the signal negligibly by folding its time rcsponse. C(l) into
S(t) resuiting in a new signal S'(t)
t S'(t) = / C(T).S(t) . (3-3)
—00
32
33
In the finai leg of the signai path, the overall measured signal,
M(t). is a convolution of S'(t) with the digitizer response, Dig(t):
t M(t) = / S'(T).Dig(t-T) dT . (3-4)
—00
Since we assume all of the above functions have a linear response
in time, the foilowing general statements hold.
If
A(t) = B(t))C:(t)
then
A(t) = C(t)HB(t) (3-5)
and if
A(t) = B(t)^ C(t) D(t)
then
A(t) B(t) :(t) > D(t) (3-6)
From the above relations for the generai functions A.B.C, and D, we
may write the measured signal, M(t) as:
M(t) = L(t))<F(t)HMcp(t) C(t)MDig(t) (3-7)
From reiations (3-5) and (3-6) we see that, mathematically, the order
of the convolution does not matter. Hence M(t) may also be written
as
M(t) = L(t) Mcp(t)HC(t)xDig(t)HF(t)
Le 11 i ng
I(t) = L(t)HMcp(t)>C(t)*<Dig(t)
allows one to write:
M(t) = I(t)»«F(t)
or equivalently,
(3-8)
(3-9)
34 t
M(t) = / I(T).F(t-T) dT . .3_10) -00
The task is to obtain the fluorescence decay function F(t) and,
from this, the various dccay times and percentage contributions of the
individuai fluorescing components. This is done with an iterative
reconvolution technique and least squares fitting (see below). First,
the instrument function has to be determined. If F(t-T) in the above
integrai is a deita-function ô(t-T), then the measured singnal M(t) is
just the instrument response I(t). Experimentaily, either a mirror or
a glass piate is used in piace of the sample, which corresponds to
repiacing F(t-T) with ô(t-T). Thus, the instrument response is
obtained and contains the contributions from the iaser pulse, MCP
temporal response, cabie contributions and preamplifier bajidwidth
(approximateiy 1 GIIz) of the fast waveform digitizer. Figure .3-2
represents a typical instrument response curve acquired experimentally
using a glass plate as the sample with the neutral density beam
splitter cube in place (see Chapter II). It is the convolution of
L(t), Mcp(t), C(t) and Dig(t) which is equivaient to the convolution
of Figure 2-3 witli the laser pulse shape.
Various deconvolution techniques have been examined by other
investigators [1,2,3]. Software has been developed by Greg Sullivan
to mathematicaily modei the fluorescence pulses for testing three of
these deconvolution techniques [4]. Tiie testing routine synthesizes
an instrument response, inciuding random Gaussian noise, and
convoiutes it with an assumed multi-exponential fluorescence decay.
The synthesized signal M(t) is then deconvoluted using the different
techniqucs to see how the calculated decay times and pre-exponential
35
coefficients compare with the known values set at the beginning. It
was found that the Fourier transform method requires intensive
operator interaction, and its ability to discern a sum of two or more
exponentials was found to be questionable. The method of moments
couid resolve single and double exponential fluorescence decays.
However. extension to three exponential decays was difficult. The
technique which was found to give the best results is the iterative
reconvoiution method, which was also found to be most satisfactory by
O'Conner e^ al. [5]. The ability to successfully deconvolute data
containing up to three different fluoresence decays, provided that
their lifetimes differ by a factor of two ajid that they each have
contributions greater than 1.5%, has l een established [6]. Results
were also tested experimentally for two and three component
non-interacting dye mixtures [6,7]. The iterative reconvolution
method assumes that the fluorescence decay F(t) from the sample is a
sum of exponential terms corresponding to emission from N individuai
fluorophores:
N
F(t) = ^ A..exp(-t/T.) . (3-11)
i = l
In general, A. is a function of wavelength. Combining this with
Equation (3-10) gives the fitting function
N
l''(t) = Y ^i'í I(T)-exp M"(t) = > A.'j I(T).exp -(t-T.)/T. —00 '-
i = l
dT (.3-12)
where A and T. are tiie parameters to be adjusted until a best fit to 1 1
the measured signal M(t) is found. This best fit is obtained wiien tiie
error sum \ is minimized, wliere
V h s j = i
) - M"(t.)
36
(3-13)
1
and n is the totai number of channels. All the 512 channels stored by
the digitizer can be used in the deconvolution routine, however, to
enhance the speed of computation, only 128 channels are generally
used. It was found that by lining up the fitting function, M°(t) with
the measured signal M(t) at 10% of the peak along the rising edge, a
large improvement in the fit results [4]. This lineup is
experimentally valid since there is a difference in the optical path
length between the acquisition of the instrument response and the
acquisition of the measured signal due to the insertion of the K399
filter and the changing of the cube from the beam splitter to the
dichroic mirror
2 The weight a. is the square of the uncertainty in the
th measurement of the i channel and is given by
a.2 = a ^ + C.M(t.) + 1 o 1
clM(t) dt " . ' ^
a^M(t) . At^ 2
dt 2 (3-14)
Here a is the baseline noise (typically 0.01 mV ). C.M(t ) the o ^
statistical counting error (^ 1 mV ), a^ the sample variance of the
time axis jitter caused by small fluctuations in tlie trigger pulse and
signal pulse rise time (a^ ~ 100 ps, depending on the number of pulses
averaged), and At {X 0.25 channels) thc fractionai channel shift used
in the time axis lineup routine. For a typical 100 mV pulse. tlie 2
maximum value for tiic third term in Equation (3-14) is 10 mV . 2
Average values for the last two terms in this equation are 2 mV and
37 2
0.25 mV . Extensive work was done in determining the uncertainties 2 ,
C7. . and more work is necessary to better understand how changes in
experimental conditions affect the various terms in Equation (3-14).
It is the author's opinion that the time shift which occurs as the
result of path length difference between the acquisition of the
instrument response and the fluorescence signal shouid be taken care
of experimentaily and not in the fitting routine. Information on very
fast kinetics may be iost in the alignment of the fitting function
with the measured signal. To the best of our knowledge, we are the
first to inciude the third and fourth terms in the uncertainty which
deal with the jitter and time axis lineup.
2 Minimizing the \ error sum is an iterative process, which
invoives incrementing the parameters A. and T. and thus successively
2 reducing \ . The method empioyed [8,9] is a combination of a gradient
2 search along the \ hypersurface and linearization of the fitting
function. Wlien far from the surface minimum, the gradient plays the
dominant roie in determining the increment chcmges. As the search
cioses on the minimum, the linearization of the fitting function
dominates. The parameter increments are given by
2n
k=l
where B. = (A.,T.), and /3, and a., are given by the relations: j ^ i i ' k jk
. , a. k 1 = 1 1
^ , c?M''(t.) aM^'^t.) r T Y 1 e" i' e'' i^ , ^ - f~ ,,-.
"jk = 2 ~ • —ôã: ôB— • h ^ jk • '^ '^
38
M^(t.) is the best fit from the preceeding iterations, A is a
weighting factor and ô ^ tlie Kronecker delta. The weighting factor
determines the relative contribution to the increments from tiie
gradient search on the hypersurface to the linearization of the
fitting function. This basic iterative reconvolution method was
developed by Marquardt [9] and has been foilowed by the author in a
new IBM/AT computer to be used with the single photon counting system
currently under developement.
The operator begins by assuming a given number of exponential
decays (1,2 or 3) and giving the computer some starting values for the
A.'s and T.'S. These input parameters are used to construct the first
fitting function M (t) by convoluting F(t) with the measured
instrument response. The program determines the change in the
parajTieters based on the above equations and forms a new set of
2 parameters. A new fitting function is then determined. The \ is
checked and if there is an improvement, tlie new values for the
parameters are kept and the proceedure again determines new ÔB . xJ
2 vaiues. If the vaiue of \ is not improved, X is incremented by an
2 order of magnitude and ôB . values are again found. Once \ improves
from the last best fit, A is reset (to 0.001). This interative
reconvolution process continues until the improvement in \ is iess
than 3% of the last best fit, at which point tiie program terminates
and displays the best fit curve with thc measured signal along with
tlie final parameters and residuals.
Time-resolved fluorescence emission spectra are obtained in 10 nm
steps by scanning the emission monochromator of the MI'V3 from 100 to
39
7.50 nm and acquiring the fluorescence decay at each wavelength. This
takes about 10 seconds (for 64 pulses, including data storage) at each
wavelength or 5 minutes total. The data reduction yields the muitipie
decay times, component spectra, and relative intensities and takes
about 4 minutes at one wavelength when fitting with a two exponential
fitting function. The total data reduction time for ail wavelengths
is 1-2 hours. If a three exponential fit is done, the total data
reduction time may increase to over 6 hours time. Programs have been
written to reduce an entire data set over an evenings time thus
increasing the efficiency of the system.
Conventionai spectra are obtained with c.w. illumination and
continuously scanning the emission monochromator. This takes about 60
seconds (depending on the scan speed selected by the operator, in this
case it is 10 nm/s), including correction for optical system response
and smoothing. Equivalent spectra are obtained by integrating tlie
time-resolved results.
For routine work, a tungsten lamp is used to determine the
spectral response of the system. The lamp is cross calibrated with a
standard lamp of a known spectral output (Eppley seriai no. ES-8319,
traceable to National Bureau of Standards reference standards, serial
numbers: F-94 and EV-8). Tliis is done by measuring the raw spectrum,
which includes the system spectral response, of both tlie tungsten and
standard lamps. The ratio of the raw tunsten spectrum T (A) to the
raw standard lamp spectrum E (A) is independent of the systcm
response. and when multiplied witli the known standard lamp spcctrum
E(A). results in the true tungsten spectrum T(A):
40
^^^^ = rJX) • ^(^) • (3-18)
The determination of the relative spectral response S(A) of a chosen
optical configuration (objective, dichroic mirror. filter. apertures.
etc.) is done routinely by measuring the raw tungsten spectrum T (A)
and dividing by the true tungsten spectrum T(A):
T^(^)
^ ^ = TJX) • (3-19)
The corrected fluorescence emission spectrum R(A) from a specimen
is then determined by taking the ratio
R (A)
^ ^ = STÃT ' ^ " ^
where R (A) is the raw fluorescence spectrum emitted from the
specimen. The above calibration procedures are built into the
software. In this way. normaiized fluorescence spectra are obtained
independently of the particular optical components chosen. The system
spectrai response corresponding to the use of a given objective,
measuring diaphragm. exit slit, and filter combination is given in
Figure 3-3. and a typicai normalized raw c.w. spectrum is given in
Figure 3-4. The upper curve of Figure 3-5 is the result of dividing
the raw spectrum by the system spectral response giving the spectrally
corrected fiuorescence emission spectrum of the specimen. The lower
curve is the smoothed version of the corrected curve; the smoothing is
done by a running average routine where the averaging window is
defined by the user along with tiie region over which smootinng is to
occur. The unsmoothed. spectrally corrected version of the curve is
stored on disk.
41
Fo test ail of the above mentioned procedures experimentally. we
measured a number of solutions containing one or more non-interacting
components with known spectra and decay times. Figure .3-6 is the
emitted fluorescence of the fluorophore POPOP in ethanol. The
measured decay time is within 10 ps of the accepted value of 1..35 ns
[10]. Many other samples were tested with similar results [6].
In order to determine the temporai sensitivity of the system, we
acquired tlie instrument response I(t) twice, once as the instrument
response and once as the assumed fluorescence (see Figure 3-7). In
theory, the decay time determined from such an acquisition should be
zero. We found that eui assumed, monoexponentiai fitting function gave
a good fit with a decay time of 7.3 ps. This result is nearly the same
as the uncertainty determined with POPOP and other solutions
(anthracene, NADH, Rhodamine etc). After determining the accuracy
and precision of our system with monoexponential decays, we went on to
biexponentiai systems. The fluorescence emitted from these binary,
non-interacting systems can be treated as a two-component exponential
function as given in Equation (3-11). Figure 3-8 represents the
result of a complete time-resolved data acquisition and reduction of a
mixture of POPOP and anthracene in ethanol. The top two graphs are
the A-coefficient spectra and a fitted fluorescence decay curve
derived from the separate solutions. The bottom graph represents the
resolved spectra and one of the corresponding decay curves acquired
from tlie mixture. It is important to note that we are able to resolve
with confidence a two-component non-interacting mixture into its
component spectra ly fitting witii a two-exponential decay function.
42
Figure 3-9 gives the A^T product spectra (equivalent to the puise
integrated spectra) of seperate anthracene and POPOP solutions. which
is aiso equivalent to the c.w. emission spectra. The bottom curve
shows the time-resolved spectra of the components along with the
mixture. Comparison of Figures 3-8 and 3-9 show that the A'T spectra
are a much more reliable means of determining the component spectra of
a mixture of non-interacting fluorophores than just the A-coefficient
spectra. This is obvious from the spurrious spike at 400 nm of the
resolved A-coefficient spectrum for POPOP in Figure 3-8. Notice also
that the structure in the anthracene spectra is better resolved in the
bottom of Figure 3-9.
When trying to fit a two-exponential system with a three
exponential fitting function, one of two things occur: either the fit
resuits in two dominant terms of high percentage and one of the
contributions is negligible, or the fit results in a redundancy of one
of the components by showing equal decay times. In other words, when
fitting with a function containing more terms than necessary, a
redundancy or negligible contribution of one or more of the components
results. For reliable results. the actual decay times have to differ
by a factor of two and the percent contribution from any component has
to be 10-20% or above.
The percent contributions of the individual components are given
by
A..T. P. = ' ' (3-21) ^ 2 A..T.
1 1 1
43
and represent the fractional fluorescence intensity of the i^^
component. The total fluorescence spectrum emitted from the specimen
is given by the sum of the components:
00
F(A) = / F(t,A) dt t=0
00
= 2 i(' ) / exp(-t/T^) dt (3-22) t=0
1
wlîich may he w r i t t e n as
F(^) = 2 ^i(^)*'^i • (3-23) i
Hence, the fluorescence intensity from the i component at A is just
F.(A) = A.(A).T. . (3-24)
This is the basis for plotting A.'T. as a function of A and 1 1
designating this the i component spectrum.
If these components are truely pure, their lifetimes will be
independent of emission wavelength as alluded to above and the
A-coefficients will vary correspondingly with emission wavelength. It
should aiso be noted that the A-coefficients are corrected for the
system spectral response. A.^T. spectra often times produce better
resuits than just A-coefficient spectra alone, especially if the
components are weak. The fitting routine compensates a large
estimation of one parameter by reducing the value of the other (A's
and T'S) for a given term in the fitting function (Equation 3-12)
resulting in an accurate result for the product, A..T, . For some
samples, sucii as coal or crude oil, tiiere are probably more than three
components making up tlie fluorescencc emitted. Hence, our thrce
component model oftcntimes gives decay times wiiich are not independent
44
of emission wavelength. We iiave found that in these circumstances,
the A.(A).T.(A) product, which corresponds to the c.w. spectrum of
this "component" (now not really well defined), produces consistent
results and sometimes the spectrum can be matched to a known spectrum
(see crude oii section).
The accuracy of the fit can be assessed from the residuals and
2 2 the reduced \^ [4], where \ ~ 1 indicates a good fit:
\ 2 ^
2
V n-j
(3-25)
where j is the number of fitting parameters to fit n channels. (n-j)
is the total number of degrees of freedom. The reduced chi squared
value of one for a good fit can be understood if one considers that
the difference between the fitted and real data should be on the order
of the uncertainty of the data, hence, the ratio of the difference and
the uncertainty shouid be one. Some of the fits presented here have
2 X values which are much less than one. This dilemma magnifies the
V
difficuity in accurately assessing tiie experimental uncertainty. It
2 should be noted that the value of \ is not important in any specific
fit as its reiative change between one, two and three exponential
2 fits. If the value of \ does not change by more than a factor of
2 three (i.e., a change in \ from 6 to 1.8) when going from a singie
to a double or a double to a triple exponential fit, then the lower
number of exponentials can be used to determine the number of
non-interacting fluorophores making up tlie sample fluorescence. This
conclusion comes from three years of experience and our work on
non-interacting mixtures.
45
The residuals, which are sho\m in the plotted fits, are defined
as the difference between the best fit M"(t) and the measured signai
M(t). Ideally, the residuals should be random and this has in fact
been observed in testing of the fitting routine with synthetic
functions when random Gaussian noise was added. However, real data
and the corresponding fits give a slight rolling in the residuals.
This rolling is most probably due to radio frequency pickup by the
eiectronics. The source of this r.f. signal is the spark gap of the
nitrogen iaser; this is verified because the use of double sheilded
coaxial cables and sheilding the MCP with aluminum foil have reduced
the effect. Increasing the number of signal averaged pulses aiso
reduces the rolling in the residuals. The quality of the fit can be
ascertained by looking at the residuais themselves. Experience shows
that the maximum residual should be less than 2% of the emitted
fluorescence peak pulse value.
The mean lifetime presented in the fitted plots is defined as:
1 P..T,
" ~ " 100
1 L (3-26)
and has proven to be useful on occasion for comparison.
46
(a) CONVOLUTION
to
TiME
(b) CONVOLUTION
>-
H-
CO
Z
TIME
Figure 3-1. Graphical description of the convolution process. (a) F(t-T) is the resuit of convôiuting a function F(t) with a delta function occurring at t=T. (b) If one has a continuous distribution of delta functions given by L(t) then convoluting F(t) with L(t) results in F'(t). F'(t) has been normalized to the amplitude of L(t) for comparison purposes.
47
TOTAL INSTRUMENT RESPONSE
37 -
30
Z) Q.
u
22
14
- 1
RISE TIME 0.64 ns
FALL TIME 1.05 ns
1/2 WIDTH 0.99 ns
1
16 20
NANOSECONDS
Figure .3-2. Total instrument response of the system. This curve is obtained by substituting a reflector (a ô-function equivalent) for the sample. The response is a convolution of the single photon response of the apparatus with the laser puise form.
48
>-t—
Z
Q LU
M I
<
ûí o z
0.8
0 .6
0 .4
0 . 0
SYSTEM SPECTRAL RESPONSE
1.0 F
0.2 -
400 500 600 700 800
WAVELENGTH (nm)
Figure 3-3. Total system spectral response S(A) for a given optical configuration (20X Rolyn objective, K399 high pass filter. 0.5 mm exit siit. 0.3 mm measuring diaphragm). This curve includes the MCP spectral response.
49
R F I E L N L A T I V
U T 0 E R N E S S I
E C T E Y N C E
o.a
0.6 -
0 .4
0 .2
400 500 500 700
WAVELENGTH /nm
800
Figure 3-4. Raw c.w. spectru R (A) of a green-yeilow resinite coai maceral.
50
24-MAR-87 09:32:16
Vp (raw) -2774 mv
PEAK AT: 519nm Q : 0.48 Qm: 1.04
AHEA PAHAMETERS: V: 21 8: 29 G: 25 Y: 17 0: 7 H: 0
SAMPLE INFORMATION
GHEEN-YELLOW HESINITE
R E L A T I V E
U T
R E L U A T I V E
F I L N T E 0
R N E S
0.8 -
0.5
0.4
0.2
400
0.8 -
0.5 -
0.4 -
0.2 -
40Q
500 600 700 WAVELENGTH /nm
800
500 500 700
WAVELENGTH /nm 800
Figure 3-5. Spectrally corrected c.w. spectrum of the same green-yellow resinite maceral as in Figure 3-4. The top curve represents the corrected spectrum and the bottom curve is the smoothed version. The spectral parameters are defined as:
V = Peak Voltage of picoammeter P
Q = Intensity at 650 nm / Intensity at 500 nm Q = Intensity at the spectral peak / Intensity at 500 nm m
The area parameters represent the area under the curve for the following wavelength intervals:
V = 420 - 480 nm B = 480 - 540 nm G = 540 - 600 nm Y = 600 - 660 nm 0 = 660 - 720 nm R = 720 - 780 nm
51
> E
33
30
23
15
a 12 16
NANOSECONDS
20
1 i -ocT-es I 1 : ; 8 : M 7
^ e í î i = '527 nm
l ^ - 0 .527 [0.527]
Q^- 1.421 mV a = 0.249 mV P o
64 PULSES
A l =
P l =
72.5
100
± 0.5
± 0
m V
%
T1 - 1.345 ± 0.011 n s
T^ = 1.346 ± 0.011 n s
1.1
- 1 . 1 RESIDUALS
POPOP IN ETHANOL
Figure 3-6. Emitted fluorescence pulse of p-bis 2-(5-Phenyloxazolyl) benzene (or POPOP) in ethanol solvent fittcd with a mono-exponential decay function. The resulting lifetime of 1.346 ns is very close to the 1..3.5 ns determined by Lakowicz [10]. whose value is judged to be within ± 0.2 ns. The accuracy of our system is better than ± 100 ps.
52
166
- 93 > E
3 CL y—
Z) O a. o 5
69 -
46
23
-1 8 12
NANOSECONDS 16 20
A^ .i - 3 7 3 nm
X^ = 0 . 7 1 3
64 PULSES
Ai - 4782 mV
Pl = 100
T^ = 0.073 ns
0.8
^ 0.0
-0.8 RESIDUALS
Figure 3-7. Self-deconvoluted instrument response assuming a mono-exponential decay. The instrument function was acquired twice, one being deconvoiuted with the o ther . The r e s u l t i n g "decay time" of 73 ps r ep resen t s the s e n s i t i v i t y of the system.
53
AN HRACENE »-COEFriCIeHT SPECTRUH
â:.m AHn 47B
UAVELENOIH Cnmi
POPOP »-COEFriCIENT SPECTRUM
iM 4nn 47B UAVELENOTH tr,i.>
mV
ANTHRACENE/POPOP HIX A-COEE. SPECTRUH
mV
_--_-;
\ MFASUREO \ Fir \ : FITTrn \ : REGION
?0 30 40
NANOSECONOS
RESIDUALS
nr.ASUREO
12 16 NANOSECONOS
RESIDUALS
MFASUHED
FiTTFD REOIOM
0 17. 10 NANOSECON S
mV
4pn *nn 470
UAVELEHOTH <r.«)
1.5
0
-1.5 RESIDUALS
Figure 3-8. Time-resolved A-coefficient spectra and corresponding decay curves for separate anthracene (T = 4.18 ns) and POPOP (r = 1.35 ns) solutions. The lower graph shows the precision of our technique in resolving components by their decay times (TI = 1.22 ns and r^ = 4.20 ns) and A-coefficient spectra. (No energy transfer takes place in this case.)
54
í iMl.SMON SPLCíRA
1 —
n _
l U
•—
_
111
A
1
l U
OC
] .0
O.U
0.6
0.'\
0.2
0.0
1.0
o.u
0.6
0.4
0.2
0.0
1.0
-.
/ /
/ >
1
1 1
A t
1 1 1
•
'• •.
"••
«
i 1 1
I 'OPOI ' •N
" • N.
N • » .
N •^ \ N \
V
1 1 1 1 1
ANTI iRACLNL
— •
• * • • • • • . .
•-•.. 1 1 1 1 7
o.u
POPOI ' •)• ANTI-IKACLNG
0.0
'.. ANTI-IRACtNE •• •. ••• ••••..
4 00 420 4 40 4 60
1
•
1
•
<>
1
DLCAY T1ML5
"C, = 1.29 nz
• • •
•
1 1 1 1
T. i - 4 .22 n.-.
• •
1 1 1 1
•
1
•
1
1 1
1
L
L
t
1»
_
-
-
T(
"C 1 - 1.27 iiG
. j * • _
11.1= 4.11 ns
I 1 i 1 \-
1.4
1.3
H 1.2
\A
\:i
).o
1/1
o > _ O \^j U l
\rt
O - T
-" i.4
1.3
1.2
4.4
4.2
4.0
3.0
H 3.6
3.4 400 4 20 440 4 60
<
WAVtLLNGTH (nm)
Figurc 3-9. Timc-resolved fluoresccnce si)cctra and dccay timcs for an anthraccnc-POPOP mixturc as a function of cmission wavclcngth. Thc top iialf of the figure shows thc rcsults for thc purc subsLanccs. the bottom half for the resolution of the mixturc. Thesc specLra (cxccpL for thc POPOP + anthracenc mixturc) arc dcLermincd by pIoLLing Lhc A -T producLs at 10 nm intcrvals. Thc spcctrum of thc mixLurc was i i
delermined by inlcgrating cach cmiHcd f Ivioicsccncc pulsc ovcr Limc and corrccting for thc systcm .speclral rosponse.
55
Literature Ci ted
1. Grinvald. A., and Steinberg. I.Z.. Biochemistry. Vol. 13. p. 5170. 1974.
2. Grinvald. A.. and Steinberg. I.Z.. "On the Analysis of Fluorescence Decay Kinetics by the Method of Least-Squares." Anal. Biochem.. Yol. 54. pp. 583-598. 1974.
3. Cundall. R.B.. and Dale. R.E., Time Resolved Fluorescence Spectroscopy in Biochemistry and Biology. Plenum Press. New York, 1983.
4. Sullivan, G.W.. "Time Resolved Fluorescence Microscopy by Pulsed Laser." M.S. Thesis, Southern Illinois University, 1983.
5. O'Conner, D.V.. and Andre. J.C.. "Deconvolution of Fluorescence Decay Curves: A Criticai Comparison of Techniques," J. Phys. Chem.. Vol. 83. pp. 1333-1343, 1979.
6. Gangopadhyay, S., Pleil. M.W.. and Borst. W.L.. "Resolution of Interacting and Non-Interacting Fluorophore Mixtures by Laser Induced Fluorescence Spectroscopy." submitted to J. of Luminescence, Feb., 1987.
7. Borst. W.L.. Gangopadhyay. S.. Pleil, M.W.. "Fast Analog Technique for Determining Fluorescence Lifetimes of Multicomponent Materials by Pulsed Laser." Presented the in Sixth SPIE Conference on Fluorescence Detection. Vol. 743. January 15-16, 1987.
8. Bevington, P.R., Data Reduction and Error Analysis for the Physicai Sciences, McGraw-Hill, New York, 1969.
9. Marquardt, D.W., "An Algorithm for Least-Squares Estimation of Non-Linear Parameters," J. Soc. Ind. Appl. Math.. Vol. 11. No. 2, pp. 431-441, June. 1963.
10. Lakowicz. J.R., Principles of Fluorescence Spectroscopy. Plenum Press, New York. 1983.
aiAPTER IV
APPLICATION TO SCINTILLATORS
Plastic and acrylic scintillators are extensively used in
particie detection because of their short fluorescence decay times.
high quantum yields and the ease in which they Ccui be shaped aná
fabricated. The transfer of kinetic energy of the detected particle
from the soivent to the primary scintillant foilows one of many
possibie paths. Since the emitted fluorescence pulse shape is
dependent not only on the scintillator but also on the source of
excitation [1-6], a detailed understanding of the energy transfer and
the contribution to the overall pulse shape from each stage of the
process is important. The overall pulse shape has been catalogued for
a number of particle, scintillator, and detector combinations, and
estimates of the contributions from the individuai energy transfer
stages have been made by various fitting techniques [1,3,7-12]. We
have measured the contribution from the last energy transfer stage of
seven common scintiliators, namely NE102A, NE104, Pilot-U, PSIO,
PS15A, BBQ90 and BBOT150, by using puised near ultraviolet laser
excitation. Our results conclusively give the fluorescence lifetime
of the final stage soiute together with its corresponding fluorescence
spectra. The measured fluorescence is caused by the transition from
the first excited singlet state to the ground state of the primary
scintiilant molecuie situated within the plastic or acrylic
scintillator enviroment. Accurate determination of these fluorescence
56
57
lifetimes effectively eliminates one fitting parameter in the overail
puise shape function. Previous work has not provided the fluorescence
lifetimes of these moiecules within the scintillators themselves.
There has been much discussion concerning fitting functions that
may describe the fluorescence emitted (or prompt response) from
unitary, binary, ternary, thick or thin scintillators following
nuciear particie excitation. The simplest system consists of only the
primary scintillant and is described by most investigators with a
unitary function (single exponentiai) [1-3,8,13]
i(t) = A exp[-t/T] (4_1)
Here it is assumed that the excitation has a negligible duration and
that the formation time of the first excited state, X as defined by
Birks [8,13], which includes ionisation, excitation, ion recombination
and vibrationai releixation, is very short compared to the fluorescence
decay time r of the primary scintillant. Aiso. the scintillator is
assumed to be transparent to the emitted fluorescence. Wlien an energy
transfer process occurs between a solvent and primary solute (or
scintillant). as is the case with binary scintillator systems. the
function which best describes the fluorescence output becomes more
complex and may be dependent on sample thickness [1]. If the system
is a liquid, it can be described well with Stern-Voimer kinetics
[1,8.10], and the emitted scintillation puise can be expressed by the
relation
i(t) a r
^ - • ' i
exp(-t/T) - exp(-t/T.) (4-2)
where r again is the fluorescence decay time of the primary solute and
T is the decay time of the solvent in the prescence of the
58
fluorescing soiute [1.2,8,10]. The energy transfer for this binary
system can be described according to Birks [8,13] as
ly- . Ix ^ ly . X'* . (4-3)
where Y represents the solvent, X the solute, and the superscripts 1
£md X represent the singlet and excited states, repectively. The
environment for binary plastic and acrylic soiid soiutions is
different from liquid solutions. Another relation is needed to
describe the emitted fluorescence. (The plastic and acrylic
scintillators are hardened liguid solutions, hence the term "solid
solution.") The binary soiid soiutions foliow Forster kinetics, which
according to Birks [5,8] results in the relation
1/2" i(t) a exp[-t/T] - exp -t/T. - 2T7(t/T ) (4-4)
where T. and r are the decay times of the solvent and soiute.
respectively, and T] is dependent on the soivent concentration. Both
Equations (4-2) and (4-4) have been used to describe plastic
scintiliator solutions with varied success [1,8,10]. In order to get
the fluorescence into a range suitable for certain photodetectors,
waveiength shifters are added to the scintillator, thus forming a
ternary soiution. These ternary systems are the least understood.
Because of the complexity of the emitted fluorescence pulse, many
different fitting functions have been tried. Two functions
successfuily employed to fit fluorescence pulses from binary and
ternary piastic solutions involve a Gaussian function muitiplied with
either a unitary fuction such as Equation (4-1) or a function similar
to that in Equation (4-2). resulting in the overall fitting functions
[1.2.14-16]:
59
i(t) = f^(t) exp[-t/T] (4_5)
and
i(t) = f^^t^^e"'/"" - e"'/''^] . (4-6)
The function f^^t) of the binary soiution in Equation (4-5) is a
Gaussian characterized by a standard deviation a, which represents the
energy transfer rate from the detected nuclear particie to the opticai
levels (or first excited state) of the solute molecule. and r is the
fluorescence decay time of the primary solute. For the ternary
piastics, fp(t) represents a Gaussian describing the rate of energy
transfer from the detected radiation to the primary solute. The
exponentiai terms in Equation (4-6) describe the energy transfer
process from the primary soiute to the wavelength shifter and the
final emission of the light from the wavelength shifter. T. is the
decay time of the solute in the presence of the wavelength shifter,
and T is the fiuorescence decay time of the wavelength shifter.
The parameter common to all of the above relations is T, the
fluorescence decay time of the primary scintillant in the presence of
the other scintillator components. The decay time r has in the past
been determined by muitpie parameter fittng of the puise emitted from
the scintillator after T-ray or nuclear particle excitation, using the
above relations convoluted with the instrument response of the
particuiar experiment. By fitting the emitted pulses with a large
number of parameters (which is necessary for high energy excitation)
previous investigators did not determine the real fluorescence decay
time T precisely nor the actual contribuLion of Lhis last stage of the
energy transfer process. We have measured the fluorescence of the
60
primary scintillant directly using near ultraviolet excitation by
puised laser, thereby eliminating the energy transfer mechanisms
present with high energy excitation but maintaining the environment of
the primary scintillant. Hence, we were able to determine r much more
accurately, and the values presented here can be used in future
studies for eliminating one important fitting parameter.
The NEÍ02A, NE104, and Pilot-U plastic scintillators were
supplied by Thorn EMI Gencom Inc., whereas the PSIO. PS15A, BBOT150
and BBQ90 acrylic scintillators were supplied by Polycast Technology
Corporation. The fiuorescence was monitored from a very small area on
the surface of the specimen. Hence, effects due to self absorption
and sample thickness did not have to be considered.
Figure 4-1 shows a typical decay piot for Pilot-U. Because we
are exciting the first excited singlet state of the primary
scintillant direcLiy, we are justified in fitting the data with a
monoexponential decay function:
A(t) = A^exp(-t/T) , (4-7)
where A(t) and A are the fluorescence intesities at time t and t=0, ^ ' o
respectively, and T is the characteristic fluorescence decay time of
the primary scintillant in its scintillator environment. This
function is the same as Equation (4-1) for unitary scinti1lators if
one also assumes, as is the case here, no self-absorption in the
samples. The accuracy of the fit was assessed from the residual and
2 2 reduced \ , where \ < l and peak residuals of lcss than 2% indicates
a good fit. The results for the other samples are given in Table 4-1.
all of which could be fitted accuratcly with a monoexponential dccay.
61
We feei that the decay times are correct within 100 ps. as was
verified by measuring the lifetimes of several substances. namely rose
bengal. PBO, anthracene, fluorescein and others, and comparing our
resuits with the known iifetimes. The reproducibility of our
experiment is weil within 100 ps. The measurements of the dependence
of the decay time on emission wavelength showed that ali decay times
(except for BBQ90) are independent of wavelength. As an example.
Figure 4-2 shows the decay time for NEÍ04 and BBQ90 as a function of
emission wavelength. NE104 is typical of the other samples showing a
tight, random distribution of lifetimes about the mean for all
waveiengths.
Figure 4-3 give the fluorescence spectra obtained with continuous
wave excitation at 365 nm. The scintillators PSiO and PS1.5A have the
same fluoresence spectra, which closely resemble those from NE102A and
NEÍ04. According to Polycast Technology Corporation, the fluorescence
we see in the two PS sampies is caused by POPOP. This is apparent
from the emission spectra. Thorn EMI Gencom Inc. was not able to
provide us with information on the primary scintillant (fluorescent
component) for "proprietory" reasons. It seems. however, that the
fluorescence detected in the NE102A and NE104 samples also is from
POPOP because of the strong resemblance of the emission spectra with
those of POPOP in solution [19]. The emission spectrum for BBQ90
matches that for BBQ obtained by Auronet [9]. and BB0T1.50 matches that
of BBOT in ethanoi [19].
The optical levels investigated in the present work are those
used in nuclear particle detection. while ihe excitaLion process is
62
different. The emission spectra of the plastic scintiilators NE102A.
NE104 and Pilot-U matched the manufacturers' spectra well. whiie the
emission spectra for the acrylic scintillators PSIO. PS15A, BBQ90. and
BB0T150 matched the spectra of POPOP in the case of the PS samples,
BBOT in the case of the BB0T150 sample and BBQ in the case of the
BBQ90 sample. As a resuit of the good match of these emission
spectra, we know that the decay times determined at those wavelengths
(corresponding to the first singlet to ground state transitions) are
those of the last step in the total energy transfer process when the
scintiliator is subjected to high energy excitation.
Our measured fluorescence decay times r are different from those
given by others (Table 4-2). The reason for these differences is in
the definition of the vaiues iisted: The decay times quoted are for
the entire pulse shape from a particular experiment, including the
energy transfer mechanism. These are not to be confused with r in the
above reiations. The uncertainty resulting from the use of a iarge
number of fitting parameters may be the reason why the investigators
who used gamma particles or high energy electrons do not give the r
values for the components of interest. Only Waynant [3] gives the
value of T. Waynant used a nitrogen laser in his determination of the
decay time of NEÍ02 and found a value of 2 ns by fitting a photograph
of a sampling osciiloscope output with a monoexponentiai function.
This value is twice as large as ours. This discrepancy is attributed
to the relative inaccuracy of Waynant's method. (NE102A and NE102 are
essentially the same type of scinti1lator; they could not be
differentiated by spectra nor by decay times.)
63
Our experience with dye soiutions teils us that the environment
of the fiuorophore can strongly influence its decay time because of a
variety of concentration and solvent effects. Birks also found
variations in the decay time of plastic scintillators as a function of
the primary scintillant concentration and composition of the solvent
(polyvinyltoluene or poiysterene; see reference [3]). These
environmental differences probably cause the variations in the decay
times measured for the various POPOP scintiilators (PSIO, PS1.5A,
NE102A, and NE104). The differences in the BB0T150 and BBQ90 decay
times as compared with the corresponding liquid soiutions also can be
attributed to environmental differences. Hence, it is important that
the fluorescence decay time of the emitting soiute be known within the
scintiliator environment. This has been accomplished in the present
work by excitation with near ultraviolet laser pulses, thus
eliminating the energy transfer contributions.
We are confident that the lifetimes determined here are those
designated as r in ail of the above relations and hence are important
in previous and future data on emitted pulses from particle detectors.
These lifetimes can be used to better understand the complex
mechanisms involved in the energy transfer of kinetic energy from the
nuclear particle to the excited singlet state of the primary
scintiilant. This may facilitate the development of better detectors
and improve the future analysis of data.
A paper on the scintillator work presented here has been
submitted to, and acccpted by Nuclear Instruments and Methods [21].
64
Table 4-1.
Scintillator
NE102A NE104 Pilot-U PSIO PS15A BBQ 90 BB0T150
Scintillator Fluorescence
Wavelength Range
.370 - 510 385 - 505 370 - 480 400 - 500 400 - 560 420 - 600 400 - 560
Lifetimes
T ± 2'0 m 1.10 ± 0.15
0.81 ± 0.03 0..58 ± 0.02 1.32 ± 0.17 1.09 ± 0.05 8.81 ± 0.81 ^ 1.23 ± 0.13
^BBQ90 exhibited a dependence of the lifetime on emission wavelength.
65
Table 4-2. Summary of
Samp1e
NE102 NEÍ04 Piiot-U PSIO PS15A BBQ 90 BB0TÍ50 BBOT in cyclohexane
(l.Og/1 concentration) POPOP in benzene
(O.ig/1 concentration) POPOP in cyciohexane
(O.lg/1 concentration)
Effective Decay Times Determined by Others
Effective decay times in ns
2.4[2],2.0[3],2.5[8],2.2[i4],2.4[17] 2.0[8],1.74[14],1.9[17] 1.36[2],1.36[10],1.38[14],1.3[17] 3.9[18] 3.9[18] 20.0[18] 28.0[18] 1.1[19]
1.26[19]
1.50[19]
Except for references [3,19], all times were determined using high energy excitation and various fitting techniques. The technique used by Waynant [3] is described in our conclusion. Berlman's fluorescence decay times were obtained by hydrogen flashlamp excitation and direct measurement of the photomultiplier output current pulse with a sampling oscilloscope [19]. The acquired signal was fitted in a manner analogous to ours, resuiting in the given decay times. Beriman's vaiues are for pure solutions at the specified concentrations.
66
1 -3 O. I -Z)
o CL
> Lli)
u
140
12
8 4
56
28
- I 0
F IT
P ILOT-U
MEASURED
2 4 6
NANOSECONDS
8 0
0 .6
^ o
- 0 . 6
- - • I
j-s^ : t^'^^m\^^ :::
• •
RESIDUALS
WAVELENGTH: 4 3 0 nm
1024 PULSES
A = 411 mV
r = 0 .583 + 0.003 ns
X^= 1.02
Figure 4-1. Emitted fluorescence pulse from a Pilot-U scintillator fitted with a monoexponentiai decay function. The resulting lifetime of 0.583 ns has a statistical error of 0.003 ns. The absolute accuracy is 100 ps as determined by direct comparison of standards.
67
co c:
LU
>-< O LLI Q
10 •
9 -
8 •
7 J
6 -
BBQ 9 0 . . • • • • • , - ^ - - . - - - ^ , . - - . . -
•
•
Tm «= 8.81 ns
1 1 1 i 1 1 1 1 1 í 1 1
i .o 4
0.9
0.8
0.7
J L » t 1
NE 104
Tm = 0.82 ns
j _ j I 1 1 1
360 4 0 0 4 4 0 480 520 560 600
WAVELENGTH (nm)
Figure 4-2. Dependence of fluourescence decay time on emission wavelength. BBQ90 is the only sample which showed a lifetime dependence on emission wavelength. The NE104 scintillator is typical of all other samples showing a tight, random distribution of lifetimes about the mean.
68
7,
O LU
M < <
o
J60 600 640
Figure 4-3. Fluorescence spectra of the seven scintiliators obtained with continuous wave excitation. The excitation source is the 365 nm line of a high pressure mercury arc lamp. The similarities between the two NE and two PS samples exist because POPOP is the common fluorophore.
69
Literature Cited
i. M. Moszynski and B. Bengston, Nucl. Instr. and Meth. vol. 158 p.Í. 1979.
2. G.F Knoll, Radiation Detection and Measurement. John Wiley, New York, 19797^
3. R.W. Waynant and R.C. Eiton, in: Organic Scintillators and Liquid Scintiilation Counting. eds.. D.L. Horrocks and C.-Tz. Peng Academic Press. New York. 1971.
4. G. D'Agostini. G. Marini, B. Martellotti. F. Mass. A. Rambaldi and A. Sclubba. Nucl. Instr. and Meth. vol. 185. p. 49, 1981
5. J.B. Birks and A.J.W. Cameron. Proc. Phys. Soc. B. vol. 69. p. .593, 1956.
6. S. Lipsky and M. Burton, J. Qiem. Phys. 31 (5), p. 1221, 1959.
7. L.M. Bollinger and G.E. Thomas, Rev. Sci. Instr., vol. .32, p. 1044. 1961.
8. J.B. Birks and R.W. Pringle, Proc. R. Soc. Eden., vol. 70, p. 233, 1971/72.
9. C. Auronet, J. Blumenfeld, B. Boso, M. Bourdinaud, P. Evard, C. Jeanney and C. Laford, Nucl. Instr. and Meth., vol. 169. p. 57. 1980.
10. F.J. Lynch. lEEE Trans. Nucl. Sci. NS-22 p. 58. 1075.
11. I.B. Berlman. J. Qiem. Phys. 33 (4). p. 1124. 1960.
12. P.B. Lyons and J. Stevens. Nucl. Instr. and Meth. vol. 114. p. 313. 1972.
13. J.B. Birks. The Theory and Practice of Scintillation Counting. Pergamon. Oxford. 1964.
14. M. Moszynski and B. Bengston. Nucl. Instr. and Meth. vol. 142. p. 417. 1972.
15. B. Bengston and M. Moszynski. Nucl. Instr. and Meth. vol. 155. p. 221. 1978.
16. B. Bengston and M. Moszynski. Nucl. Instr. and Meth. vol. 117. p. 227. 1974.
70
17. Thorn EMI Gencom Inc. product literature (Gregory Kapp).
18. Polycast Technology Corporation product literature (John Lee).
19. A.B. Berlman. Handbook of Fluorescence Spectra of Aromatic Moiecules. Academic Press. New York. 1971.
20. W.L. Borst, M.W. Pleil, G.W. Sullivan, J.C. Crelling and C.R. Landis, in: New Applications of Analytical Techniques to Fossil Fueis. American Chemical Society. Division of Fuel Chemistry. New York. April 13-18. (1986) vol. 31 (1) pp. 7-16,
CIIAPTER V
APPLICATION TO GEOLOGICAL SPECIMENS
Coal
Fluorescence microscopy has been a usefui tooi for the
characterization of organic materiais in coals and rocks [1].
Carbon-rich coal is composed of microscopic organic equivalents of
minerals called macerals. We studied macerais under near ultraviolet
excitation and are the first to investigate the fluorescence emitted
from these organic compounds in the time domain. By applying the
present technique. we have shomi that the maceral fluorescence can be
modeled adequately with a sum of exponential decays. Hence. more than
one fluorophore is present.
In order to determine the usefulness of time-resolved
fiuorescence analysis as an extension of conventionai fluorescence
analysis of organic materials found in coals and rocks, two sets of
samples were studied. One set was a series of shale samples at
different levels of maturation from the New Albany Formation from the
Illinois Basin. While maturation trends of this material in the
Illinoes Basin have been studied using vitrinite reflectance (a
standard measure of maturity) and qualitative and quantitative
fluorescence techniques [2.3,4], nothing previously was kno\Yn of the
time-resoived fluorescence properties of these (or any) sampies. The
other set was a series of samples from the lower Kittanning seam from
Pennsylvania and Oliio, which has a more extended range in rank, from
71
72
.47 to 1.01 vitrinite reflectance (from high-volatile bituminous C to
medium-voiatile bituminous).
Two primary fluorophores were found in alginite macerais from the
New Aibany Shale Group of the Illinois Basin. Alginite macerais are
generally yeiiow to orange in coior, microscopic in size (10-100 im)
and are believed to originate from prehistoric aigea. The data
acquisition consisted of 21 decay curves, one tai<cen every 10 nm over
the fiuorescence emission range of 450-650 nm for each alginite
macerai investigated. Each decay curve acquired was individually
deconvoluted with an assumed two exponential fitting function
resulting in the determination of the component A-coefficients and
corresponding decay times. Figure 5-1 represents one such acquired
curve and its corresponding fit. Figure 5-2 represents the results of
fitting the 21 decay curves acquired from one sample at 10 nm
intervais; A., T. and P. are plotted as a function of emission
wavelength. The coefficients A.(A) make up the component
A-coefficient spectra. Since the decay times remained relatively
constant over the emission wavelength range, which is expected for two
non-interacting fluorophores, our two component model is justified for
this sample set, and the spectra represent the individual component
fluorescence, which make up the total fluorescence. The A-coefficient
spectra were averaged over 45 alginite macerals covering a limited
range of maturity and the resulting spectra are given in Figure 5-3.
The dotted lines reprcsent twice the sample variancc; hence, there are
definitely two separate componenLs conLributing to the fluorescence.
Each individual A-coefficient spectrum was normalizcd to unit area
73
prior to averaging (we were interested only in the general shape of
the spectrum and not the absolute intensity which is dependent on
sampie size as well as aperture settings). The average spectra were
then normalized to unit height for comparison. The blue component of
Figure 5-3 has a lifetime in the subnanosecond range (0.2 ns). while
the red component iifetime is in the nanosecond range (3.0 ns).
Figure 5-4 represents a typical c.w. emission spectra of an alginite
maceral. Figure 5-5 shows that the percent contribution to the total
intensity (P.) averaged over the entire emission range of each
fluorescing component. The percent contribution has a noticeable
correlation with sample maturation (or rank). The maturity or rank of
the sampie was determined by standard vitrinite reflectance
measurements. which ranged from 0.4 to 0.62 in vitrinite reflectance.
With increasing rank. the two primary fluorophores of alginite
macerals change only in their percent contributions (relative
intensities). and the individual component spectra remain the same. as
also indicated by the small sample variance in Figure 5-3. It seems
that as maturation occurs. one fluorophore may be transforming into
another type or both fluorophores are changing into non-fluorescing
products at different rates.
The Kittanning coais have a wider rank range (0.47 to 1.01
vitrinite reflectance) and an abundance of different maceral types.
All of the time-resolved data was again fit with a two exponential
fitting function. This Lwo-componenL analysis was performed on Lhree
of the niain maceral types. Figure 5-6 shows the unique distribution
of decay times as a function of imiccral type. The short component
74
iifetime T^ is shortest for sporinite. followed by alginite (New
Aibany) and fluorinite. The longer component r^ is shortest for
alginite, foilowed by fluorinite and sporinite. The averaged
A-coefficient spectra for two of the three macerai types are given in
Figure 5-7 (aiginite component spectra are given in Figure 5-3). Only
the fluorinites showed a spectral seperation of the quality in Figure
5-3, hence the two-component analysis is probabiy valid here. The
other maceral type shows larger deviations in the A-coefficient
spectra, which is a direct result of both the complexity of the system
(this maceral is most probably comprised of many fluorophores) and the
quality of the data (only 64 pulses were averaged for each decay
curve). Some of the two component lifetimes showed a correlation with
rank suggesting that our two-component model may be inadequate.
however, the fluorophore environment is also changing resuiting in a
different non-radiative decay rate which changes the observed
component lifetimes. (This is analagous to the scintillators where
the lifetimes depended on the type of solvent and its concentration.)
There may also be some energy transfer mechanisms invoived or there
may be a distribution of iifetimes present. (The fluorescence emitted
from a single protein fluorophore, for example, can be represented
with a distribution of lifetimes since there is probably a
distribution of configurations the fiuorophore may be in [5,6].) Even
with these complications, the technique has shown that the resolved
component spectra from this two component model can be used to
identify the macerals. Fluorinite and sporinite spectra differ
significantly. For fluorinitc, the short lifctime spectrum peal<s in
75
the biue. while the long lifetime spectrum peaks broadly in the
yeilow. Conversely. sporinite spectra are more broad; the short
lifetime component peaks near 5.50 nm (yeliow) and the longer lifetime
component peaks in the green. This relationship between the decay
times and spectra of sporinite is opposite to that of alginite.
thereby distinguishing these important macerals and providing a basis
of identification of those petrographicallly troublesome constituents
of sedimentary rocks. Resinite fluorophores also peak in the
yeiiow-orange region of the visible spectrum but the shorter iifetime
has a larger contribution from the blue.
Our resuits for coal groups indicate that temporal fluorescence
analysis is an extension of conventional coal characterization
techniques by measuring the fluorescence lifetimes for various
macerals. The observed change in percent contribution of the
fiuorescing components with increasing rank represents the first
measurement of this kind in coal petrology. The resolution of the
totai fluorescence emitted from a geologicai specimen into its
component fluorophore spectra and the tracking of the component
spectra through a range of maturity levels has never been done before.
Cude Oil
We also have used our technique to characterize crude oil samples
from petroieum deposits in Alabama. California. Texas. and Venezuela.
The technique is capable of analyzing three dominant fluorophores in
the samples. Figure 5-8 shows Lhe Lime resolved specLra and
fluorescence decay times of a Texas crude oii. Also shown are the
76
continuous wave excitation spectrum and pulse integrated spectrum for
the sample. Characteristic decay times and spectra of the short lived
component (T^ = 1.68 ns) and a longer lived componcnt (T„ = 18.9 ns)
resemble standard reference spectra and lifetimes of anthracene and
pyrene. respectively [7]. These component spectra were found by
plotting the product A.-T. as a function of wavelength in 10 nm steps.
The spectra are the actual component spectra. obtained by integrating
the fitting function over time. Large deviations in r resulted for
the short lived component beyond 500 nm; these deviations are most
probably caused by the component's low fluorescence intensity. Figure
.5-9 shows three plots of API index (proportional to the inverse of
specific gravity and a standard geological measure of maturity) versus
the component lifetimes of all the oil sampies. The lifetimes of all
three components decrease with decreasing API index. This decrease in
the lifetimes is probably due to the increasing percentage of
asphaltenes. which act as fluorescence quenchers. The quenchers ailow
the excited fluorophores to return to the ground state via more
numerous nonradiative paths. The decrease in API from condensates to
heavy crude oils is related to the decrease in maturation levei.
Hence the lifetime of the components can give direct information about
the maturity of these oil samples. The peak of the c.w. spectra is
also shifted from red to blue with incrcased maturation levels for
these samples.
77
241
193
m V 144
96
48
m V
- 4
18-MAR-85 15: 15:10
A MEASURED / \ FIT 1 l
\ : ; FITTED \ : ; REGION
1 \
1 \
/ ^-~~~^
0 2 4 5 8 1
NANOSECONDS
RESIDUALS
0
1
'^em
' " p ^
54
Al
A2
P1
P2
Tl T2
^ .
=
—
500 nm
0 . 5 5
9 .97
PULSES
=
=
=
=
=
=
1188 .8
51
7 4 . 8 7
2 5 . 12
0 . 2 4 2
1.897
0 . 5 5 8
[ 0 .
mV
+
+
+
+ +
+
55]
O o = 0
18 .2
1.5
0 .B4
0 . 8 4
0 . 0 0 5
0 . 0 3 2
0 . 0 1 8
243
\ 1
mV
% o/ /o
ns
ns
ns
•p.V
iOILOlLl ALGINITE
Figure 5-1. Fiuorescence pulse emitted from an alginite macerai after puised laser excitation. The pulse is the convolution of the instrument temporal response and the actual fluorescence of the sample. The exit monochromator was set at 500 nm. The low \ value and a peak residuai of less than 2% indicate a good fit.
78
2 140aA ALG.2
10000 r
.000
A^(mv) ^oo
10 .
10 r
T^ (ns)
100
Pi(X) 50 •
0
FLUDRE5CEi\CE DECAY PARAMETERS
450 550 550
WAVELENGTH/nm
750
A2
Tí T2
iii
Pl
P2
Figure 5-2. Example of time-resolvcd fluorescence from an alginite coal maceral. The wavelength dependence of the A-coefficients. fluorescence decay times. and percent contributions of the fluorescing constituents are given.
79
NEW ALBANY SHALE RANK SERIES
C 0 E F F I C I E N T
400 500 600 700
WAVELENGTH (nm)
LEGEND
800
2 X SamplB Varlanca
Average Spectrum
TOTAL NUMBER F SAMPLES: 45
WAVELENGTH RANGE: 450 - 550 nm
Figure 5-3. Resolution of the composite fluorescence spectrum emittcd from alginite macerals of New Albany shale. Two time-resolved component spectra are obtained (normaiized to unit height). The •'blue" component has a lifeLime in the sub-nanosecond range (0.2 ns). and the "red" component in the nanosecond range (3.0 ns). The individual component spectra remained the same for 45 macerals. but Ihe percent contribution of cach component depended on sample maturi ty.
80
19-FEB-85 t4108I30
Vp (raw) -5000 mv
PEAK AT: 536nm Q : 0.56 Qm: 1.07
AREA PARAMETERS: V: 16 B: 32 G: 31 Y: 20 O: 1 R: 0
SAMPLE INFORMATION
A3 lOILOlLl ALGINITE
R E L A T I V E
R E L A T I V E
400 500 600 700 WAVELENGTH /nm
8 0 0
0 . 8
0 . 6
0 . 4
0 . 2
4 0 0 5 0 0 6 0 0 7 0 0
WAVELENGTH /nm
8 0 0
Figure 5-4. A typical continuous wave emission spectrum of an alginite coai macerai with the corresponding spectral parameters
81
r fíW ALOANY O I L SMALE RANK S E n i E ES
r = 0.5743
PERCENTAGE 1
91
81
70
59
43
38
Slope = ^3.858
0.4 0.4-1 0.43 0.53
nEFLECTArsCE IX)
0 . 5 8 0 . 6 2
r = - 0 . 6 6 5 9 S l o p e = - 5 9 . 4 3 5
PEnCEriTAGE 2
61
50
40
10
0.4 0 . 4 4 0 . 4 9
f lEFLECTANCE C
0 . 5 3 0 . 5 0 0 . 6 2
Figure 5-5. Perccnt contribution to thc total intcnsity avcraged over the cntirc cmission range as a function of sample maturity. The percent contribution of each component has a noticeable correlation with sample maturation (vitrinite reflectance).
82
QJ ^ W
P^ : 3 o CJ
o o
o IS w O' w o^ \M
w >
H
H-I
w
20 -1
10
0
0
0
SPORINITE
T^ = 0 .18 ns
T r
T^ = 4 .13 ns
0 1 2 3 4
10 -
FLUORINITE
T-j = 0.32 ns ^2=3.17 ns
íli 0 1 2 3 A
20 -
10
mT-j_ = 0.21 ns ALGINITE
^2=2.15 ns
=] P=^
0 2 A 5
=i .
7
-1 1 1 1
6 7
7
FLUORESCENCE DECAY TIME / ns
Figure 5-6. Distribution of fluorescence decay times in coal macerals. Two characteristic groups of decay times are discernible for each maceral type.
83
c
F. F F I C I A H T
400
C L) F; F F I c [ A tl T
500 GOO 700
WAVELENGTH (nm)
800
KITTANNING HANK GEfUES
LEGENU
i P O n i N I T E :
2 X Sampl t í V a n a n c e
A v e r a g c S p e c t r u m
TOTAL NUMUER OF SAMPLES: 3G
WAVELENGTfl flANGE: 450 - G50 nm
400 500 GOÛ / 0 0
WAVELLMGll-l (nm)
000
KITTANr i IMG IIA'i;-; GL f l I LS --- ILUU11INITL5
TOFAL NUMULÍI Or GAMPLES: 34
Figure 5-7. Resolution of composite fluorescence spectra of the two major maceral types found in the Kittaiining coals. The fluorinite macerals were the only type which could be modeled well with two components. The other maceral shows larger standard deviation in the component spectra indicating the complexity of the system.
84
&-• M LO 2; w [-1
M
CU
u w u íf)
D.
o T
1.0
0. 0
0. 6
0. 4
0. 2
0. 0
12
10
PULSE INTEGRATED SPECTRuM
c.w. EMISSION SPECTRUM
0
/'''' '~*\ TIME - RE S 0 L VE E - T =18. 9ns/ \
// T.-,-^ T3ns ^\(
1 1 1 1 1
) SPECTRA
DECAY TIMES
0 n n n o o ' u " u «•
0 o o
^2
W
0
u
X
n " « A "
• . • • •
• .
1
300 420 460 500 540 500 300 420 460 500
WAVELENGTII (nm)
540 500
Figure 5-8. Fluorescence emission spectra and lifetimes from a Texas crude oil. The top graph shows the c.w. emission spectrum and pulse-integrated data. The lower graph contains three time-resolved component spectra and the corresponding decay times found by fitting the fluorescence pulses with a thrce exponential decay functon.
85
a ^
<:
u Q
20
16
12
8
1
2
0
-10 0 10 20 30 AO 50 60
API-INDEX
Figure 5-9. Dependence of the three resolved fluorescence decay times (see also Figure 5-8) on API index for several crude oii and condensate samples. (A higher API index means greater sample maturity). A direct relationship is indicated between the component lifetimes and maturity.
86
Literature Cited
1. Borst, W.L.. Pleil. M.W.. Sullivan. G.W.. Landis. C.R.. and Creiling. J.C, "Laser-Induced Fluorescence Microscopy of Coal Macerals and Dispersed Organic Material." in New Applications of Analytical Technigues to Fossil Fuels. American aiemical Society. Division of Fuel Chemistry Vol. 31. No. 1. pp. 7-16, New York. 1986.
2. Barrows. M.H.. Cluff. R.M.. AAPG Memoir 35: Petroleum Geochemistry and Basin Evaluation. p.lll. (1984).
3. Barrows. M.H., Cluff. R.M., Harvey, R.D., Proc. 3rd Eastern Gas Shale Symp., p. 85, (1979).
4. Cox. G.E.. M.S. Thesis. Southern Illinois University at Carbondale. (1985).
5. Dowben. G.. and Lin. G. "Use of Nanosecond Pulse Fluorometry for Lhe Study of Protein Structure," Sixth SPIE Conference on Fluorescence Detection, Vol. 743, Jan. 15-16, 1987.
6. James, D.R.. Liu. Y.S.. Siemiarczuk. A., Wagner. B.. and Ware, W.R. "Recovery of Underlying Distributions of Lifetimes from Fluorescence Decay Data" Sixth SPIE Conference on Fluorescence Detection, Vol. 743. Jan. 15-16. 1987.
7. Berlman, A.B.. Handbook of Fluorescence Spectra of Aromatic Moiecuies. Academic Press, New York, 1971.
aiAPTER VI
APPLICATION TO CRIMINALISTICS
Application of our fast analog technique lends itseif very weli
to the study of forensic materials. We have applied our technique to
hair, fibers and also common surfaces encountered in fingerprint
detection. The microscope allows us to select the specific areas
where we can then determine the c.w. fluoresescence and time-resoived
spectra.
Fibers are a common material in criminal investigations and must
often be distinguished from one another. We have applied our
technique to fibers, which look alike under the microscope and have
the same fluorescence spectra (Figure 6-1). The fluorescence pulses
emitted from two such similar fibers are given in Figures 6-2 and 6-3.
The results of fitting the pulses with a bi-exponential fit indicate
that for the white sock fiber of Figure 6-2. the fluorescence decay is
essentially mono-exponential (P. = 98.4%). Figure 6-3 gives the
resuits of using a bi-exponential fit to the fluorescence pulse
emitted from a string fiber. Clearly. there are two primary
components making up the total fluorescence. each having a
characteristic lifetime. We believe that the shorter component of 1.0
ns (T ) is the same for both samples. By looking at one emission
wavelength we have been abie to distinguish between the two fibers
based on fluorescence decay times. fibers which otherwise would be
considered identical.
87
88
We have also applied our technique to determine the fluorescence
lifetimes of materials commonly encountered in fingerprint detection.
Laser detection of latent fingerprints is oftentimes difficult on
surfaces such as wood and brown paper due to the large background
fluorescence from the surfaces. We measured the lifetimes of these
background materials so that Dr. Roland Menzel couid determine the
feasibility of using a gated imaging technique for fingerprint
contrast enhancement [6-1]. If the background has a short lifetime
while the desired image (fingerprint) has a long lifetime. then all
one needs to do is gate the detector enabling it to operate oniy after
the background fluorescence has decayed (ordinary photographic
techniques fai1 due to the short time scale). An image intensifier
tube with the proper gating electronics is ideal for this use and an
order of maginitude increase in contrast is expected [6-1].
The materials investigated and corresponding lifetimes are given
in Table 4. Both blue excitation (373 nm using BPBD dye) and green
(508 nm using Coumarin 500 dye) excitation were used. Green
excitation is preferred here since most fingerprint laboratories use
argon-ion or Nd-YAG (frequency doubled) lasers which produce green
light. It was found that fingerprints treated with
cyanoacylate/rhodamine 6G. which is often used for staining
fingerprints. have a lifetime of about 3.4 ns (see Figure 6-4). much
longer than most of the materials in Tabie 6-1. Hence. this proposcd
gated-imaging technique would work quite well.
89
rable 6-1. Fingerprint Background Materials
Material
Wood
Wood
Cardboard
Cardboard
Cardboard
Leather (rough)
Leather (smooth)
Brown Envelope
White Paper
Ye11ow Paper
Exci tation Wavelength
(nm)
373
508
508
508
373
373
373
373
508
373
Emission Wavelength
(nm)
587
587
599
559
585
540
439
434
588
539
Lifetimes (ns)
Ti = 0.568 T2 = 2.067
Ti = 0.573 T2 = 2.124
Ti = 0.999
Ti = 0.917 T2 = 3.234
Ti = 0.160 T2 = 1.502
Ti = 0.100 T2 = 1.841
Ti = 0.164 T2 .= 2.280
Ti = 0.462 T2 = 3.139
T, = 1.110
Ti = 0.451 T2 = 2.086
Percent
Pi P2
Pi P2
Pi
Pi P2
Pi P2
Pi P2
Pi P2
Pi P2
Pi
Pi P2
= 44.53 = 55.47
= 45.73 = 54.27
= 100.0
= 97.21 = 12.79
= 59.85 = 40.15
= 63.21 = 36.79
= 69.70 = 30.30
= 63.28 = 36.72
= 100.0
= 73.06 = 26.94
90
H
CO
w
W u w co w
o
1.0
0 . 8 -
0 . 6 -
0 . 4 -
0 . 2 '
0 . 0 400 500 600
WAVELENGTH/nm
700
Figure 6-1. Fluorescence spectra emitted from a cotton string fiber and a fiber from a white sock. Both fibers could not be distinguished under the microscope nor could they be separated on the basis of their c.w. fluorescence emission spectra.
91
591
472
354
m V
235
117
-1
m V
L
11.5
-11.6
MEASURED FIT
FITTED REGION
0 4 8 12 16 20 NANOSECONDS
A1 A2
Pl P2
Tl T2
=
=
=
=
=
=
1350.4
2 .2
98.42
1.57
0.G45
7.952
± 6.5
± 0.8
± 0.75
± 0.75
± 0.004
± 2.532
mV mV
/o
%
ns
ns
T„ = 0.957 ± 0.074 m
ns
RESIDUALS
Figure 6-2. Fluorescence pulse emitted from a white sock fiber fitted with a bi-exponential fitting function. The fluorescence decay is essentially mono-exponential (Pi = 98.-1%).
92
413 F
331
248
m V
165
B2
-1
m V 7.4
-7.4
MEASURED FIT FITTED REGION
4 8 12 16 20
NANOSECONDS
Al A2
Pl P2
Tl T2
=
=
=
=
=
—
753.5
127.1
75 .18
24.81
1.008
1.999
+
+
+
+
+
+
18.1
20 .2
3.15
3.16
0.019
0 . 1
mV mV
%
%
ns ns
T^ - 1.254 ± 0.076 m ns
RESIDUALS
Figure 6-3. Fluorcsccnce pulse emitted from a cotton string fiber fitted with a bi-exponential fitting function. The fluorescence decay is clearly made up of two components. It is believed that the shorter component of 1.0 ns (T^) is due to the same fluorophore for both the cotton and sock fibers.
93
Literature Cited
Menzel. E.R.. Pleil, M.W.. Gangopadhyay. S., "Enhancement of Fluorescent Fingerprints by Time-Resoived Imaging." Sixth SPIE Conference. Voi 743, Jan. 1.5-16. 1987.
CONCLUSION
The present teclmique has great utility for the characterization
of fluorescing microscopic substances. In criminalistic applications
the ability to differentiate between virtually alike fibers has been
shown. The fluorescing constituents of coal and crude oils can now be
resolved. The actuai fluorescence lifetimes of the primary
scintillant in seven commercially availible scintillators have also
been found and can be used to better understand the complex mechanisms
involved in the transfer of the kinetic energy from the nuclear
particle to the excited singlet state of the primary scintillant. thus
facilitating the development of better detectors and improving the
future analysis of data.
Since our preliminary studies on source rocks and crude oils have
sho>vn the existance of very short. picosecond constituents. it was
necessary to construct a new system employing a single photon counting
technique. A synchronously pumped, tunable dye laser system has been
chosen. purchased. installed and tested. and wiil be the new puised
excitation source. The output laser puises are at 368 nm and have a
full width at half maximum of less than 4 ps. This short pulse width
will excite the shorter components more efficiently than the 500-700
ps pulses of the nitrogcn pumped dye laser. Even though the
excitation efficiency for the longer lived components will decrease
wi th the use of the siiorter pulses. these components are in general
more intense. hence. they should still be resolvable (they can also be
94
95
resolved with the fast analog technique). The average power of the
new laser system at a repetition rate of 4 MHz is routinely between 4
and 5 mW (1-1.2 nj/pulse). At repetition rates of 400 kllz. a pulse
energy of over 3 nj has been measured. The computer hardware for the
new system consists of a PCA board which converts out IBM-AT personal
computer into a muitichannel analyzer. This hardware has been
installed. tested and linked to an EG&G Ortec time to emiplitude
convertor (TAC). The TAC is linked to a fast photodiode trigger
source and the MCP via two constant fraction discriminators and a
linear ampiifier. A total instrument response of 103 ps has been
measured. however. implementation of better triggering techniques and
better timing units should improve the instrument response to about 75
ps. Based on experience with the oid technique and the deconvolution
routine. it is hoped that a precision of less than 10 ps in the
lifetime determination can be achieved, hence allowing the resolution
of fast events other than fluorescence. A Fortran deconvolution
routine based on Marquardt's routine has been written ajid tested with
synthetic data. Further testing is planned to determine the
theoretical limitations of the routine, its precision as a function of
instrument response half width, channel number and total counts will
also be determined. After the theoretical limitations have been
determined, the system will be experimentally tested using the same
reference materials as with the old system (rose-bengal, POPOP.
rhodamine 6G, etc).
Current research is aimed at better understanding the origin of
oil. its maturation process and its basic chemical structure. It is
96
hoped that the chemistry of the individual fluorophores may be found
so that the fluorescence source can be determined and traced through
the evolution of the oil or source rocks. To better understand the
mechanisms involved in fluorescence emission, other, simpier systems
are being investigated. Pure polymers treated with dye rotors are
studied to determine the rotational properties of the rotors in the
poiymer environment. The observed fluorescence lifetime is a function
of the free volume availible to the rotor, hence, the more volume
available, the more the dye is allowed to rotate and the shorter the
observed lifetime. These rotor studies will help us understand the
fluorescence properties of dye stained insulators which have been
damaged through electric discharges. Possible time-resolved
luminescence studies on semiconductors have also been discussed. The
applications of both our new and old systems to all of these various
materials indicate the utility of the technique to materials research.
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