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Femtosecond laser studies of biological systems
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Content
FEMTOSECOND LASER STUDIES OF BIOLOGICAL SYSTEMS
Darin Joseph Files
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CHEMISTRY)
May 1996
Copyright 1996 Darin Joseph Files
UNIVERSITY O F SO U T H E R N CA LIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA S0OO7
This thesis, written by
under the direction of h.l.£.....Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
Dtan
Date..Al?I?.L
THESIS COM M ITTEE
Acknowledgments
First I w ould like to express m y sincerest appreciation for Professor Larry
D alton's su p p o rt for the past several years. H e has been an im m ense source
of guidance an d w isdom , both in science and life. I w ould also like to thank
him for sending m e to France for the International C onference on N onlinear
Optics. It w as truly an experience of a lifetim e that I w ould never have
dream ed could come true. I w ould also like to thank him for his stim ulating
and often funny group m eetings. W ithout a doubt he has firm ly established
th at chem istry is an integral com ponent for the technology of tom orrow .
Special thanks to the D alton group m em bers, both past and present. In
particular I w ould like to m ention A aron H arper for synthesizing several
com pounds and helping m e w ith som e chem ical techniques, and for
Sham m y=Shajing for being so hard w orking. H e w as truly a source of
inspiration and interesting conversations. O ther g roup m em bers I w ould like
to acknow ledge include Rima Ghosn, VounSoo Ra, and A ndrea H ubbell.
This group, along w ith the others, had som e fun times. Thanks also for m y
u n d erg rad u ate co-w orker Julie M uche, and Joe Larsen for helping m e w ith
the DFWM m easurem ents of the phthalocyanine sam ples. Also, thanks to
Dr. Fritz Strohkendl for the other m easurem ents as well as the w isdom he
shared w ith me.
Finally, m any thanks to m y fellow m em bers of the W estm inster and
Irvine Vanpools. I w ould have never had m ade it through g rad u ate school
com pletely sane w ithout them. In particular, I w ould like to acknow ledge
Tyson, DeAnn, Bob, Leon, Suddha, Pat, Steve(s), Michel, and Barbara. They
truly m ade the ride hom e fun.
iii
Table of C ontents
CHAPTER 1............................................................................................................................. 1
Im plem entation of a Ti:Sapphire Fem tosecond Laser for U ltrafast N onlinear
O ptical M easurem ents
1.1 Introduction.................................................................................................................. 1
1.2 Research A genda....................................................................................................... 3
1.3 Fem tosecond Laser Fundam entals........................................................................ 4
1.4 Fem tosecond Pulses Produced by Ti:Sapphire Lasers.....................................7
(A) Ti:Sapphire Gain M edium ......................................................................7
(B) Shutter............................................................................ 8
(C) Kerr Lens.....................................................................................................8
(D) D ispersion C om pensation..................................................................... 9
1.5 Practical Considerations for Mira 900 Fem tosecond Laser..........................12
(A) Oscillator Cavity................................................... 12
(B) Pum p Laser Source and Specifications.............................................13
(C) Tunability................................................................................................. 14
(D) M odelocking............................................................................................14
(E) Dispersion C om pensation................................................................... 14
(F) M aintenance and Unexpected Problem s.........................................15
1.6 References.............................................. 16
CHAPTER 2........................................................................................................................... 18
R egenerative Am plification of Fem tosecond Pulses by the C hirped Pulse
T echnique
2.1 Introduction............................................................................................................... 18
2.2 Design C onsiderations for Fem tosecond A m plifiers.....................................19
2.3 C hirped Pulse Am plification................................................................................20
(A) Pulse Stretcher........................................................................................ 21
(B) Optical Isolator........................................................................................23
(C) Am plifier C avity.................................................................................... 25
(D) Pulse Com pressor..................................................................................28
(E) Phase Com pensator...............................................................................29
2.4 References....................................................................................................................... 31
iv
CHAPTER 3........................................................................................................................... 33
Fem tosecond DFWM of the Photosynthetic Bacteria R hodobacter Sphaeroides
3.1 Introduction..................................................... 33
3.2 O scillatory Features of Photosynthetic Reaction C enters of
Bacteria....................................................................................................................... 34
3.3 Bacteriochlorophyll dom ing as a com ponent in Vibrational
C oherence.................................................................................................................. 35
3.4 Rational for studying the PRC w ith Fem tosecond DFWM......................... 36
3.5 Experim ental Protocol, M easurem ents, and D ata...........................................37
3.6 References........................................................................ 41
CHAPTER 4........................................................................................................................... 44
Fem tosecond DFWM of M etal-Substituted Chlorophyll-a and
Bacteriochlorophyll-a
4.1 Introduction................................................................................................................ 44
4.2 Experim ental Rational for M etal-Substituted C hlorophyll-a.....................44
4.3 Isolation and Purification of C hlorophyll-a from Spinach.........................47
4.4 M etal Substitution reactions of C hlorophyll-a................................................ 50
4.5 Fem tosecond DFWM M easurem ents................................................................. 55
4.6 Vibrational C oherence in isolated C hlorophyll M olecules?.......................60
4.7 References.................... 60
CHAPTER 5............................................................................................................................62
Light A ctivated Catalytic Antibodies and Bacteriorhodopsin
5.1 Bacteriorhodopsin Background............................................................................ 62
5.2 Photoactive Catalytic A ntibodies..........................................................................64
5.3 References....................................................................................................................65
v
4
5
6
6
10
10
11
12
21
25
29
30
37
38
39
v i
List of Figures
H eisenberg U ncertainty Relation
G aussian Function Used to fit a Fem tosecond Pulse
H yperbolic Secant Function used to fit a Fem tosecond Pulse
Frequency C ontent of a Fem tosecond Pulse Centered
at 760 nm
Pulse Broadening experienced by a 100 fs pulse after
consecutive passes through a 2 cm KDP crystal
D ipersion C om pensation Scheme used in C oherent
M odel 900 Fem tosecond Laser
Taylor Series Expansion for D ispersion C om pensation
Oscillator Cavity in Coherent M ira M odel 900
Fem tosecond Laser
Stretcher C onfiguration used in CPA
O ur A m plifier Layout Schematic
Com pressor Design used in CPA
C urrent O u tp u t from o u r Fem tosecond A m plifier
Visible Spectrum of R hodobacter Sphaeirodes
Initial pre-filtered Fem tosecond DFWM Signal
Post-filtered Fem tosecond DFWM Signal
Fig. 4.1 Chemical Structures of C hlorophyll-a and
Bacteriochlorophyll-a 45
Fig. 4.2 A bsorption Spectrum of Bacteriochlorophyll-a
in Acetone 46
Fig. 4.3 Acid A ddition to Chlorophyll-a to form Pheophytin-a 51
Fig. 4.4 M etal Insertion Reaction 53
Fig. 4.5 C hlorophyll-a UV-Vis Spectrum 54
Fig. 4.6 C opper C hlorophyll UV-Vis Spectrum 54
Fig. 4.7 Nickel C hlorophyll UV-Vis Spectrum 54
Fig. 4.8 Zinc C hlorophyll UV-Vis Spectrum 54
Fig. 4.9 Fem tosecond DFWM Signal of C opper C hlorophyll 55
Fig. 4.10 Enlargem ent of Figure 4.10 55
Fig. 4.11 transient grating results 56
Fig. 4.12 Phthalocyanine sam pled exam ined by Fem tosecond
DFWM at 738 nm 58
Fig. 4.13 UV-Vis Spectrum of Phthalocyanine Sam ple 58
Fig. 4.14 Fem tosecond DFWM Signal from Pthalocyanine 59
Fig. 5.1 Binding Pocket Schematic of Bacteriorhodopsin 62
Fig. 5.2 W ater-Soluble H apten C hrom ophore for MOPC 315 64
vii
Chapter 1
Implementation of a Ti:Sapphire Femtosecond Laser for Ultrafast
Nonlinear Optical Measurements
1.1 Introduction
For the past few years, a m ajor goal of o u r research in nonlinear
spectroscopy has been to define m echanism s of optical nonlinearity in quasi
one and tw o dim ensional conjugated oligom ers and polym ers.1 Some
illustrative exam ples of quasi-one dim ensional organic m aterials include
polyenes, cyanines, polyacetylene, and polydiacetylene, w hile two
dim ensional conjugated system s include the organom etallic ring structures
such as porphyrins and phthalocyanines. These type of organic system s
typically possess a strong electron-phonon coupling, w hich m eans that there
exists a very close relationship betw een the electronic and geom etric structure
of the m aterial. Any m odification in the electronic structure follow ing a
resonant photoexcitation process results locally in a fast (picosecond to
subpicosecond) structural relaxation. This, in turn, can lead to the form ation
of nonlinear excitations such as solitons, polarons, bipolarons, or polaron-
excitons. M oreover, fast m ultiphoton de-excitation pathw ays, w hich
originate from strong electron-phonon coupling, can lead to recovery times
that in som e instances can be on the order of picoseconds.2
U ltrafast structural relaxation associated w ith strong electron-phonon
coupling m ay find potential applications in nonlinear optical (photonic)
devices. For exam ple, nonlinear optical phenom ena observed for these
organic system s m ay form the basis for all-optical devices such as optically
bistable sw itches and nonlinear directional couplers.3
1
The suitability for these m aterials for nonlinear optical devices requires a
large m agnitude for the respective nonlinear process (i.e., the nonlinear
dependent refractive index associated with the third-order optical
susceptibility, anc* a sm all signal attentuation arising from the linear
and nonlinear (m ultiphoton) absorption.3 H ow ever, in spite of the fast
response times exhibited by these organic m aterials, the optical nonlinearity
figures of m erit for device applications are relatively sm all, due either to
losses from linear absorption (one photon) or m ultiphoton resonances in the
w avelength region w here the m aterial is initially tran sp aren t W hen large
nonlinearities are reported, their origins are usually traced to m echanism s
involving resonant enhancem ent.4 W hile resonant processes generally have
larger nonlinear susceptibilities, the response tim es are typically slow er
because real electronic excitations occur. The lifetim e effect of the electronic
excited state can lim it the cycling tim e betw een successive nonlinear
interactions. In addition, considerable absorption loss occurs to the optical
beam w hen exciting the nonlinear optical m aterial on resonance. O n the
o ther hand, nonresonant processes, which involve the instantaneous
polarization of the pi-electron cloud, occurs on fem tosecond tim e scales.2
Therefore, the nonlinear response is extrem ely fast. Since non-resonant
processes are described by virtual excitations, the signal beam avoids
attenuation. U nfortunately, except for the best polyacetylene sam ples, the
third order nonlinear susceptibilities of these m aterials, m easured far from
single and m ulti-photon absorption, have proven to be too sm all for device
applications.2 Therefore, increasing attention has been given to
m easurem ent protocols w hich perm it definitions of m echanism s of optical
2
nonlinearity and to schem es exploiting optical nonlinearities derived from
resonant photoexcitations.1
1.2 Research Agenda
O ur research goal was to study the dynam ics of photoexcited states of
quasi-one and two dim ensional system s as well as to characterize the
tem poral behavior of both the real (nonresonant) and im aginary (resonant)
com ponents of the various contributions to the nonlinear susceptibilities,
and relate these values to structure and interm olecular interactions. To
achieve our research objective, w e incorporated a fem tosecond laser system
for ultrafast nonlinear optical m easurem ents. M easurem ent protocols that
w e w anted to integrate into our system included the following: Degenerate
Four W ave Mixing (DFWM) w ith appropriate phase sensitive detection to
separate the the real and im aginary com ponents of the third order
susceptibility tensor, N ondegenerate Four W ave Mixing, and tw o w ave
m ixing experim ents such as pum p-probe and D ynam ic Kerr Effect
experim ents, w hich m easure, respectively, the im aginary and real
com ponents of the third order susceptibility. W ith these experim ental
protocols, w e w ould be able to establish nonlinear optical properties such as
sign, w avelength dependence, tem poral dependence, concentration
(interm olecular interactions), and the tensorial nature.1
D uring the time w e incorporated our laser system for nonlinear optical
m easurem ents, \ve encountered several obstacles that prevented and delayed
us from achieving som e of our original goals. To a large extent, our
fem tosecond laser system has been, and continues to be, an integral yet at
times an unfulfilling com ponent of our research efforts. Because of the
am ount of effort that was p u t into setting up our fem tosecond laser system , a
overview of its developm ent, operation, and applications will be given.
Since several review articles are available on fem tosecond pulse generation
and am plification, only key concepts and relevant equations will be included
w hen necessary. Particular em phasis will be placed on the experience and
know ledge I gained while assisting in the developm ent of our fem tosecond
laser system.
1.3 Femtosecond Laser Fundamentals
In order to understand the technology behind our fem tosecond laser
system , one needs to be fam iliar w ith the frequency bandw idth of a
fem tosecond optical pulse, as well as the m athem atics that describes it.
An optical pulse of fem tosecond tim e duration is com posed of a coherent
superposition of m any optical frequencies that interfere constructively in a
short interval of tim e and space, and destructively everyw here else. From
fourier analysis, a light pulse of fem tosecond tim e duration m ust consist of a
large spread of optical frequencies, the exact m athem atical relation of w hich is
given by the H eisenberg U ncertainty relation.5
AEAt^/2 ai)
The tim e duration of a fem tosecond pulse is determ ined experim entally
by an autocorrelation. In this process, a fem tosecond pulse is split into two
4
paths, w ith one path having a variable length. At the point of intersection,
the tw o pulses interact w ith the KDP crystal, and through nonlinear wave
m ixing, create a second harm onic signal at the phase m atching condition.
W ith an appropriately positioned detector, the intensity of the second
harm onic signal is m onitored as a function of pathlength difference betw een
the tw o pulses. Because of the nonlinear w ave m ixing process of the second
harm onic signal generated, m axim um intensity is obtained w hen the tw o
pulses have m axim um tem poral and spatial overlap. This occurs w hen there
is no pathlength difference betw een the tw o pulses. As the pathlength
changes, how ever, the intensity of the second harm onic signal is reduced.
Following through several pathlength differences, both positive and
negative, leads to the desired pulse shape w hich can then be fitted to any one
of tw o m athem atical functions: the G uassian or the H yperbolic Secant, the
m athetm atical relation of w hich are given below.
Gaussian Pulse Shape
I(t) = exp[-(41n2)(t/xp)2]
£ ° ‘8 “
< 7 5
| 0.6 -
(D
£ 0 . 4 -
rt
a >
tr
0 . 2 -
0.0
-5 0 50 150 *150 -100 0 1 00
Time (Fem toseconds)
Figure 1.2 Gaussian Function used to fit a Femtosecond Pulse
Hyperbolic Secant2 Pulse Shape
I(t) = sech2(1.76t/TD)
1,0 -I
^ 0-8-
| 0.6 -
©
| 0.4 -
®
£E
0 .2 -
- 1 5 0 -1 00 -5 0 0 50 100 150
Time (Fem toseconds)
Figure 1.3 Hyperbolic Secant Funtion used to fit a
Femtosecond Pulse
A lthough both functions can be used to fit to the autocorrelation function,
and thereby determ ine the pulse duration, w e and several others typically use
the latter function to m easure pulse duration. W ith the pulse duration
exeperim entally determ ined, w e can next check to see the required spread of
optical frequencies required to produce a pulse of such short timescales.
1 . 0 -1
0.8 -
tn
| 0.6 -
a
I °*4 -
a >
C O
0.2 -
0.0
720 740 760 780 800
Wavelength (nanometers)
Figure 1.4 Frequency Content of a Femtosecond Pulse
Pulse Centered at 760 nm
6
1.4 Femtosecond Pulses Produced by Ti:Sapphire Lasers
In order to generate an optical pulse of fem tosecond tim e duration, several
criteria m ust be met. First, a sufficient num ber of optical m odes m ust be
present to satisfy the H eisenberg relation. Second, a coherent superposition
m ust be m aintained betw een these optical m odes in order to create the
desired transform lim ited pulse. A nd finally, net gain m ust be m aintained
for all optical com ponents of the pulse. W hat follows next is a brief
description of how our comm ercial fem tosecond laser achieves the above
criteria.
(A) Ti:Sapphire Gain Medium
The three m ost fundam ental com ponents of our com m ercially available
fem tosecond laser are the resonator cavity, the optical frequency gain
m edium for self-sustaining the feedback, and an appropriate pu m p source to
provide energy to the gain m edium . The resonator cavity, for exam ple,
determ ines two key output param eters for our fem tosecond laser. First, the
cavity length defines the optical w avelengths (longitudinal or laser m odes)
that are allow ed to oscillate w ithin the laser cavity. A longitudinal or laser
m ode is an optical field distribution that reproduces itself after one round trip
inside the laser cavity.6 A nother laser param eter by the cavity is the
repetition rate or tim ing betw een consecutive pulses. In order to generate
enough optical m odes to form a fem tosecond pulse, a broad gain bandw idth
m aterial is needed. In our laser, the gain m edium is a T itanium doped
Sapphire crystal. Am ong the properties that m ake Titanium doped Sapphire
7
an ideal m aterial for fem tosecond pulse generation are its large fluorescence
gain bandw idth (660nm-1180 nm), high saturation fluence and dam age
threshold, and its relatively long excited state lifetime of 4 m icroseconds.7
(B) Shutter
O ne of the basic requirem ents for pulse production is that sufficient gain
be available for all the laser m odes that contribute to pulse form ation,8
How ever, the gain bandw idth curve of Ti:Sapphire is frequency d e p en d e n t
As a result, the m odes w ith higher gain extract m ore of the available energy
and suppress the other m odes. The net result, in w hich only a few m odes are
allow ed to lase, is called continuous w ave "CW" operation. U nder CW
operation the pow er of the output does not vary w ith tim e.9 In order to
initiate pulse production, som e m echanism m ust be present to create a
w indow of opportunity for the relatively low gain m odes w hile, at the sam e
time, the high gain m odes experience net loss. D istributing the gain over a
w ider range of laser m odes is achieved by an optical shutter. Located w itin
the laser cavity, the shutter w orks by changing the am ount of glass that the
beam transverses. Because of the different phase delays experienced by the
optical m odes, the standing wave condition is altered, and for a brief instant, a
larger num ber of m odes are lasing than norm al.9
(C) Kerr Lens
A lthough the optical shutter allows m ore of the m odes to laser, the result
is only tem porary. A nother m ethod m ust also be present to m aintain the
gain for all m odes that contribute to pulse form ation. To m aintain net gain
for the pulse and net loss for the CW background, Ti:Sapphire lasers use the
intensity-dependent self-focusing effect of the Kerr Lens, along w ith an
appropriately positioned aperture w ithin the laser cavity. The Kerr Lens
Effect describes the nonlinear, intensity dependent refractive index change
experienced in the gain m edium during pulse operation. U nder these
conditions, the Kerr Lens, present in the gain m edium during pulse
propagation, causes the beam to self-focus. Thus, at certain locations w ithin
the cavity (i.e., the position of the aperture) the m odelocked beam is narrow er
than the CW beam . W ith sim ple adjustm ent of the slits’ w idth, the narrow er
m odelocked beam is allow ed to oscillate uninterrupted w ithin the cavity
w hile the m ore diffuse beam associated w ith CW operation experiences losses
in the form of clippings along the edges. Therefore, the m odelocked beam is
allow ed to extract m ore of the energy stored in the gain m edium , and thus
overtake the CW m ode as the m ost stable form of operation.9
(D) Dispersion Compensation
W hile the optical shutter, K err Lens, and aperture com bine to initiate and
sustain gain for the m odelocked beam , a pair of prism s is used to m aintain
the phase coherence betw een the lasing m odes. As the pulse oscillates w ithin
the cavity, the individual frequency com ponents experience different
propagation speeds as they through the gain m edium . As a result, phase
coherence is dim inished and pulse broadening results.6 If left uncorrected, the
pulse will continue to broaden until the intensity of the pulse is insufficient
to form the Kerr Lens. (See Figure)
0 .8 -
0 . 2 -
0.0
800 600 400
time / 10 fs
200
Figure 1.5 Pulse Broadening experienced by a 100 fs pulse after
consecutive passes through a 2 cm KDP crystal
At that p o in t the resulting o u tp u t w ould resem ble noise since the m odes
w ould be superim posed independently.5 To alleviate the frequency-
dependent phase shift experienced by the pulse as it travels through the gain
m edium , a pair of prism s is arranged such that the faster red com ponents of
the pulse experience a longer path distance than the slow er blue
com ponents.9
Components of
positively chirped
pulse
Red
Red and Blue
Components of
Pulse Together
Blue
Figure 1.6 DispersionCompensation Scheme used in
Coherent Model 900 Femtosecond Laser
Ideally, this w ould cancel the frequency dependent phase shift, w hich is called
G roup Velocity D ispersion. H ow ever, there are also higher o rd er term s that
also contribute to pulse broadening. In particular, the linear change in G roup
Velocity D ispersion, w hich is know n as T hird O rder D ispersion, has recently
been show n to be the prim ary lim itation to u ltrash o rt p u lse generation in
Ti:Sapphire based lasers.10 The third an d higher order term s originate from a
Taylor Series Expansion around the central frequency of the pulse as given
below :
cp(co) = o(coo) + < t> '( coo) (co-coo) + t&"(coo)(co-coo)2/2! (1.7)
+ ^..(coOKco-coO^/s!
The derivatives identify the follow ing pulse properties: the first derivative
defines the group velocity of the w avepacket, the second governs the rate at
w hich the frequency com ponents of the w ave packet change their relative
phases, an d the third determ ines the linear change in G roup Velocity
D ispersion.5
A lthough the gain ban d w id th of Ti:Sapphire can theoretically su p p o rt a 3
fem tosecond pulse, im perfect third, and to a lesser extent, fourth o rder
dispersion com pensation has prevented this pulse d u ration from being
obtained. C urrently, the shortest pulses obtained from Ti:Sapphire lasers are
just below 10 fem toseconds.5 Tw o key advances have allow ed researchers in
the field to m ove closer to the gain b an d w id th lim it of Ti:Sapphire. First,
certain glasses used for the prism s have better dispersion com pensation
properties than others. For exam ple, fused silica and BK-7 are the tw o m ost
cited prism glass m aterials that are used in fem tosecond lasers for phase
com pensation. In essence, choosing the right glass m aterial for the prism
pairs can reduce the pulse duration. By using this technique, one group
11
reduced their pulse duration from 60 to 30 fem toseconds. A nother im portant
im provem ent, again m ade by the sam e group, w as the use of m ore highly
do p ed (x5) Ti:Sapphire crystals. A n increase in the concentration of T itanium
allow s m ore of the light from the A rgon ion laser p um p source to be absorbed
in a shorter crystal. A shorter crystal leads to less m aterial, and thus less
dispersion com pensation to be corrected for.5
N ext, a m ore practical view point of o u r fem tosecond laser will be given.
Topics to be covered include the laser cavity layout, operation, m aintenance,
an d unexpected problem s w e encountered shortly after w e started to set up
the system for nonlinear m easurem ents.
1.5 Pracitical Considerations for Mira 900 Femtsecond Laser
(A) Oscillator Cavity
Below is the optical schem atic for our fem tosecond laser cavity (C oherent
M ira M odel 900).
M 7
Figure 1.8 Oscillator Cavity in Coherent Mira
Model 900 Femtosecond Laser
12
As depicted, the cavity length is determ ined by the distance between m irror 1
(the output coupler) and m irror 7. Also show n are the shutter and aperture
that combine to produce fem tosecond pulses, as well as the prism pair which
m aintains pulse fidelity. The com ponent labelled BRF refers to the
Birefringem ent filter w hich is used for w avelength tuning.9
(B) Pump Laser Source and Spcecifications
D epending on the w avelength and desired output pow er, the fem tosecond
oscillator can be pum ped w ith 6.5 to 10 W atts available from the A rgon Ion
laser w hich operates under m ultiline configuration. For o u r laser system , w e
have the A rgon Ion laser adjusted to provide approxim ately 8 W atts of pow er
to the Ti:Sapphire crystal. C oupling betw een the pum p laser beam and the
ThSapphire crystal is achieved through a pair of m irrors located w ithin the
fem tosecond laser cavity. Both m irrors can be adjusted to control the vertical
and horizontal overlap of the pum p beam w ith the Ti:Sapphire crystal.
A nother function of the pum p m irror set is to flip the polarization of the
A rgon Ion beam. As m entioned previously, Ti:Sapphire has an absorption
profile that prefers horizontal over vertically polarized light.11 Because the
beam output from the A rgon Ion laser is vertically polarized, the m irror set is
arranged such that the polarization is flipped. Thus the m irror set enables the
Ti:Sapphire crystal to be pum ped w ith horizontally polarized light.
Param eters that can be adjusted on our fem tosecond laser include the
tuning range, pum p pow er intensity, slit w idth, and the am ount of dispersion
com pensation.9
13
(C) Tunability
A lthough the gain b an d w id th is relatively large, the tuning range for the
oscillator is lim ited in p art d u e to the w avelength d ep en d en t reflective losses
that arise from the cavity m irrors. Three set of m irrors are available for short
w avelength (720-810 nm ), m iddle w avelength (8.00-900 nm ), and long
w avelength (890-990 nm ) tuning. W ithin a given set of m irrors, an even
n arro w er w avelength band is selected through the use of a birefingem ent
filter.9
(D) Modelo eking
As described earlier, a m echanism m ust be used to initiate fem tosecond
pulse production. To create a w indow of opportunity for fem tosecond pulse
production, the CW m odelocked sw itch is turned to the "m odelocked"
position. By doing so, the starter, depicted in the laser cavity schem atic,
begins to oscillate rapidly. As a result, the laser m ode condition changes such
that a larger num ber of m odes are allow ed to oscillate. The ap ertu re is
adjusted until stable pulses are form ed.
(E) Dispersion Compensation
N ear transform lim ited pulses can be obtained through the u se of the
prism pair. In order to com pensate for GVD, prism #2 is laterally displaced as
depicted in the laser cavity schem atic. As the fem tosecond p u lse passes
through the prism #1, the pulse's frequency com ponents are dispersed.
14
Therefore, the frequency com ponents of the pulse experience a different
phase lag. The net result is a near transform lim ited pulse.
(F) Maintenance and Unexpected Problems
D uring the early part of o u r fem tosecond laser set-up, w e encountered
three different problem s that degraded the m odelocking perform ance and
lim ited the usefulness of the laser to our experim ental goals of ultrafast
nonlinear optical m easurem ents. O ne of the first problem s w e encountered
w as poor m odelocking stability. We noticed that the m odelocking
m echanism w ould start to deteriorate after a few hours of start up. A t the
time, the only rem edy w as to readjust the pum p m irrors and som etim es
realign the optical com ponents of the oscillator. Even thorough cleaning of
the optical com ponents lead to im proved but only tem porary relief in
m odelocking stability. Clearly, the m odelocking instability w as preventing us
from perform ing any m eaningful nonlinear optical m easurem ents. It w as
quite ironic, therefore, how w e stum bled into one source of m odelocking
instability. A pparently the distance betw een the pum p laser and the
fem tosecond oscillator w as a critical factor. A fter returning from a building
evacuation, w e noticed vastly im proved m odelocking stability. This
suggested to us that m odelocking m ay be sensitive to m echanical a n d /o r
electrical disturbances or fluctuations present in a occupied building. To test
the m echanical disturbances on m odelocking quality, w e m oved the A rgon
Ion pum p laser closer to the fem tosecond oscillator. From such a sim ple
m anuever, w e w itnessed a substantial im provem ent in m odelocking
stability.
N ext we noticed that an unusual am ount of dust w as collecting on the
optics. Two rem edies w ere used to solve this problem . First, w e purchased a
full canopy, along w ith side shields, to cover both lasers. W e also purchased a
fan w ith an air filter in order to pum p dust-free air into the enclosure. W hile
the canopy helped considerably, the air filtered system appeared to stir up
m ore dust. In order to keep the oscillator cavity relatively dust-free, w e
connected a nitrogen gas outlet to the cavity, thereby creating a positive
pressure differential.
These tw o solutions w orked quite well in solving o u r earlier m odelocking
instability. U nder current operating conditions, the laser system needs only a
few m inutes before stable m odelocked pulses are form ed. W hile w e found
sim ple rem edies to the m odelocking instability, the second problem ,
insufficient pulse energy for nonlinear m easurem ents, has proven to be both
m ore difficult and time consum ing. This aspect of our research will be
discussed in the next chapter.
1.6 References
1. L. R. Dalton, L. S. Sapochak, and L. Yu, T . Phvs. C hem .. 1993, 97, 2871-2883
2. J. L. Bredas, C. A dant, P. Tackx, A. Persoons, and B. M. Pierce, Chem . Rev..
1994, 94,243-278
3. D. C. Rodenberger, J. R. Heflin, and A. F. Garito, N ature. Septem ber 12,
1992, 359,309-311
4. V. S. W illiams, S. M azum aadar, N. R. A rm strong, Z. Z. Ho, and N.
Peygham barian, T . Phvs. C hem .. 1992, 96,4500-4505
5. H. C. Kapteyn and M. M. M urnane, Optics and Photonics N ew s. M arch
1994,20-28
16
6. W. L. W eaver and T, L, Gustafson, Spectrochimica Acta Rev.. 1993, 15, 527-
579
7. F. P. Strohkendl, D. J. Files, and L. R. Dalton, T , Opt. Soc. Am. B., May 1994,
11, 742-749
8. E. P. Ippen, U ltrafast Phenom ena VIII. 1993, 55,155-159
9. O perator's M anual for the Coherent M ira M odel 900 Laser
10. J. P. Zhou, G. Taft, C. P. H uang, M. M. M urnane, H. C. Kapetyn, and I. P.
Christov, Optics Letters., A ugust 15,1994,19,1149-1151
11. P. F. M oulton, T.Opt.Soc.Am.B., 1986, 3,125-131.
17
Chapter 2
Regenerative Amplification of Femtosecond Pulses by the Chirped
Pulse Technique
2.1 Introduction
Femtosecond laser technology progressed rapidly during the time we
began to incorporate our system for nonlinear optical m easurem ents. Several
im provem ents in Ti:Sapphire fem tosecond lasers led to shorter pulse
durations and higher pulse energies.1 W hile our Ti:Sapphire laser produces
pulses w ith sufficient time resolution, the energy per pulse, or peak pow er, is
in general too low for nonlinear optical m easurem ents.2-4 H ow ever, several
linear and nonolinear m easurem ents can still be m ade and have been
reported in the literature w ith the sam e laser that w e have.
In order to increase our pulse energy, we incorporated a regenerative
am plifier into our fem tosecond laser system. Besides increasing the pulse
energy, a regenerative amplifier also increases the tuning range through
second and third harm onic generation, nonlinear processes w hose
efficiencies increase w ith pulse energy.2 An even broader tuning range can be
achieved from high peak pow er femtosecond pulses through the use of w hite
light continuum generation, which requires pulse energies on the order of
m icrojoules.5
Because of the im portance of high peak pow er pulses to our goal of
studying m echanism s of ultrafast optical nonlinearity, a review of the
regenerative amplification process will be given next.
2.2 Design Considerations for Femtosecond Amplifiers
Fem tosecond optical pulse am plifiers are designed w ith sim iliar
considerations as their ultrafast laser counterparts. For example, both are
designed to m inim ize spectral dispersion that arises w henever a fem tosecond
pulse transverses optical com ponents such as lenses, prism s, crystals, etc.6
Also, the total am ount of dispersion is m inim ized by reducing the m aterial
path length in the am plifier cavity. This includes using a shorter, m ore
highly doped crystal, and by substituting refractive optics w ith reflective
m irrors w herever possible.6 As with the fem tosecond laser cavity, state-of-
the-art fem tosecond regenerative am plifiers also include a prism pair set that
enables phase com pensation for both second and third order dispersion.
Thus, fem tosecond pulses w ith m icrojoule energies are now com m onplace.6'
10 A nother common feature betw een fem tosecond lasers and regenerative
amplifiers is the choice of gain m edium . For the past few years, Ti:Sapphire
has been the prim ary choice to am plify fem tosecond pulses generated by
Ti:Sapphire fem tosecond lasers. Ti:Sapphire is also an excellent am plifying
m edium because of its high energy storage capacity, its long excited state
lifetime, and its high therm al conductivity.1
W hile TkSapphire fem tosecond lasers and Ti:Sapphire fem tosecond
am plifiers share sim iliar design principles, they also exhibit som e im portant
differences. Key am ong them is the use (or lack thereof) of nonlinear effects.
Earlier we saw that the Kerr Lens Effect was used in conjunction w ith a
properly positioned aperture to initiate fem tosecond pulse production. The
Kerr Lens present in Ti:Sapphire under m odelocked operation served as an
optical switch that enabled the high intensity fem tosecond pulses to self-focus
19
through the aperture. Thus, the pulses w ere allow ed to propagate through
the cavity w ithout losses. However, the m ore spatially diffuse CW beam
suffered losses (clippings) from the aperture, and was subsequently
extinguished. After one round trip, a portion of the pulse was transm itted
outside the laser cavity via the output coupler. O n the other hand, the
am plifier is designed to have the pulse oscillate w ithin the cavity for several
round trips. This enables the pulse to extract as m uch of the available energy
deposited in the Ti:Sapphire crystal. W ith each successive pass through the
Ti:Sapphire crystal, the pulse energy grow s exponentially until the stored
energy is depleted. Beyond a m axim um point, the dam ping or loss factor
present inside the cavity (i.e., losses due to reflections and scattering) begins to
dom inate. W ith each successive pass beyond the m axim um energy point, the
pulse experiences losses by a constant factor.1 1
2.3 Chirped Pulse Amplification
As the pulse energy increases, so too does the efficiency of nonlinear
effects associated w ith high peak pow er fem tosecond pulses. Because of
potential detrim ental nonlinear effects associated w ith high peak pow er
fem tosecond pulses during amplification, such as beam steering, gain
saturation, spectral hole burning, beam distortion, and possible crystal
dam age, fem tosecond pulses are tem porally stretched to picoseconds prior to
amplification. The technique by which fem tosecond pulses are stretched to
picoseconds, amplified then recom pressed back to fem toseconds is called
C hirped Pulse Amplification, CPA.1-3-12' 13 CPA consists of five components:
a pulse stretcher assembly, an isolator, a regenerative am plifier cavity, a pulse
20
com pressor, and a pulse com pensator. Each com ponent's function and
configuration will be review ed next.
(A) Pulse Stretcher
O ne of the first steps used in the CPA technique is to tem porally stretch
the fem tosecond pulses to picoseconds. Fem tosecond pulses are stretched to
picoseconds by a grating pair assembly arranged in an antiparallel
configuration1' 3_4' 7>13_14 as show n below:
2 f
Figure 2.1 Stretcher Configuration used in CPA
In contrast w ith the prism pair assem bly in the laser cavity, which created a
negative group velocity dispersion,15 the grating pair is arranged to reduce
the pathlength for the red com ponents of the pulse and thus create a positive
group velocity dispersion. In turn, this leads to a frequency-dependent phase
shift for the optical com ponents of the w ave packet. W hen the com ponents
recom bine, a tem porally stretched pulse is created.16
Red chirping a fem tosecond pulse is carried out as follows. First, a pulse is
directed tow ard a grating which subsequently disperses the optical
21
com ponents of the pulse as show n in the figure above. From the frequency
dependent angle of diffraction, the blue com ponents of the pulse are
dispersed closer to the grating's norm al. Along w ith the antiparallel
configuration, the frequency-dependent diffraction angle creates a shorter
pathlength for the red com ponents of the pulse as depicted above.
After the pulse is dispersed by the first grating, the beam becomes
elongated w ith the long axis parallel to the optical bench. Next, the elongated
beam experiences a frequency dependent pathlength difference that arises
from the tilt of grating #2. From here the spatially seperated com ponents are
dispersed again by grating #2. The elongated beam is next directed toward a
m irror which is used to retrace the beam s path along the stretcher assembly.
As the beam retraces its path, the optical com ponents experience another
frequency-dependent phase shift, again arising from the tilt of grating #2.
Thereafter, the elongated beam is recollim ated by the lens pair as the beam is
directed back tow ards grating #1. Finally, the tem porally broadened pulse
leaves the stretcher assembly. To conserve space, w e use the m irror-folded
configuration which requires only one grating and lens.
Four passes are used in our set-up to convert fem tsoecond pulses to
picoseconds. Thus, w ith this arrangem ent, a stretching factor on the order of
a thousand is achieved.4 - 8'13 W ith our system, for example, we seed the
am plifier w ith 130 fem tosecond pulses generated from our oscillator. The
pulse train entering the stretcher assem bly is first attenuated in order to avoid
burning the holographically encoded, gold coated diffraction gratings which
have groove densities of 2000 lines per m illim eter.11
As indicated from the stretcher configuration, the input (unstretched) and
o utput (stretched) beam s are coincident. Therefore, after only one pass
22
through the stretcher assembly, the stretched pulse w ould retrace its path back
to the oscillator. This can lead to negative feedback which disrupts the
stability of modelocking. In order to avoid feedback, the collim ating lens is
vertically displaced above the optical axis. The vertical offset serves two
purposes. First, it allows us to m ake several passes through the stretcher
assem bly. W ith each pass we can exam ine in sequence the entire stretching
process. Each pass through the stretcher assembly leaves a distinct trace that is
characterized by its beam shape (elongated or oval) and vertical position,
either above or below the optical axis. W ith the aid of an IR viewer, we can
see alternating patterns of "lines" and "dots" on both the grating and folding
m irror. As a result, the beam ’ s alignm ent and spatial profile can easily be
assessed and if needed, properly adjusted. More im portant the veritcal offset
allows us to easily identify the final stretched pulse and redirect it tow ard the
isolator w hich will be covered next.
(B) Optical Isolator
After a fem tosecond pulse is extended to picoseconds, the beam is next
directed tow ard the amplifier. Here, the pulse experiences up to a m illion
fold gain in energy.3- 9 However, as was the case for the stretcher, the
incom ing (pre-am plified) and outgoing (post-am plified) pulse are coincident.
Therefore, a m ethod m ust be used to distinguish betw een the two beams.
The isolator assem bly serves this function in two steps sum m arized next.
The first function of the isolator is to rotate the polarization of the
unam plified pulse from horizontal to vertical. Next, the vertically polarized,
stretched pulse is coupled into the regenerative am plifier assem bly via a Thin
23
Film Polarizer, TFP, w hich has polarization-sensitive transm ission and
reflection properties. Once inside the am plifier cavity, the pulse experiences
another 90 degree polarization rotation (vertical to horizontal). A fter the
pulse finishes its final pass, the polarization is rotated by another 90 degrees,
horizontal to vertical. The vertically polarized, am plified pulse is then
coupled out of the am plifier cavity by the TFP. Finally, the pulse passes
through the isolator w hich m aintains its vertical polarization. A calcite
polarizer then directs the beam tow ard a half-w ave plate and the com pressor
assem bly.
The isolator assem bly consists of three com ponents. In sequence from the
stretcher to the am plifier, they include a calcite polarizer, an achrom atic half
w ave plate, and a Faraday rotator. The stretched pulse first encounters the
calcite polarizer w hich is adjusted for m axim um transm ission of the
horizontally polarized beam . Next, the beam passes through an achrom atic
half-w ave plate w hose fast and slow optical axeses are arranged such that a
single pass results in a counter-clockw ise polarization rotation by 45 degrees.
Finally, the beam is rotated by another 45 degrees (counterclockwise) as it
passes through the Faraday rotator. The Faraday rotator is adjusted to rotate
polarized light counterclockw ise by 45 degrees regardless of the direction of
light propagation. As w e shall see shortly, this is exactly opposite to the
polarization rotation in the achorm atic half-w ave plate in w hich the rotation
(clockwise or counterclockw ise) depends on the direction of propagation.
24
(C) Amplifier Cavity
The next step is to couple the vertically polarized pulse inside the
am plifier cavity. This is achieved by a thin film polarizer w hich exhibits high
transm ission for horizontally polarized light (98%) and high reflectivity for
vertically polarized light (85%) from 700 to 950 nm .14 The regenerative
am plifier assem bly consists of a Pockel's cell, a m ode-m atched resonator
cavity, a Ti:Sapphire crystal, and a Nd:YAG p um p source as show n below:
Ti:Sapphire
130-180 fs,
770 nm, 76 M Hz
- 0 Grating
Joule Meter
PC
TFP
M3
Ti:Sapphire
M2
Pol X/2
Nd:YAG
Figure 2.2 Our Amplifier Layout Schematic
25
The Pockel's cell consists of a KDP crystal with a sol-gel coating. Its function is
to rotate/ by tim e-gated voltage steps, the polarization of the pulse as it is
coupled into and out of the am plifier assem bly.1 1 The am plifier cavity
consists of concave end m irrors w ith relatively small radii of curvature of 1.5
m (M l) and 0.75 m (M3) in order to m aintain a large m ode volum e at both
the Ti:Sapphire crystal and the Pockel's cell and thereby m inim ize any
associated nonlinear effects. For convenience of space, the am plifier is folded
once by a planar m irror (M2). The length of the am plifier is identical to that
of the laser. As w ith the Ti:Sapphire crystal in the laser, the Ti:Sapphire
crystal in the am plifier cavity is brew s ter cut, 10 m m long, and doped w ith
0.15 w eight percent of titanium oxide.1 1 The Ti:Sapphire crystal is
longitudinally pum ped with a frequency doubled output from a Nd:YAG
laser (Model Surelite from Continuum ) at 532 nm , 20 H z repetition rate, 5 ns
pulse duration, and approxim ately 13 mj of energy. The total am ount of
energy available by the Nd:YAG laser (125 m j) can be adjusted by a half-wave
plate and a polarizer.1 1
The regenerative amplification process works as follows. First, the optical
axis of the KDP crystal inside the Pockel's Cell is adjusted such that a static
quarter w ave birefrigence is given After the first pass through the Pockel's
Cell, the veritcally polarized pulse experiences a 45 degree rotation. The pulse
is then reflected back into the Pockel's Cell by the am plfier cavity end m irror,
labelled M l. A second pass through the Pockel's Cell gives a horizontally
polarized pulse. N ow the pulse can transm it through the TFP and enter the
am plifier cavity. The pulse is now capable of extracting energy from the
TkSapphire crystal. Up to this point, however, the Ti:Sapphire crystal in the
am plifier cavity has not been pum ped by the Nd:YAG laser. After two passes
26
through the Ti:Sapphire crystal, the pulse is redirected back tow ards the
Pockel's Cell. A nother round trip through the Pockel's Cell leads to a
veritcally polarized pulse. Therefore, w ithout applied voltage, the injected
pulse becom es circularly polarized after one round trip through the am plifier
cavity. As the vertically polarized pulse arrives at the TFP, it is reflected back
tow ards the isolator assembly.
In o rder to increase the num ber of passes through the am plifier cavity, a
quarter-w ave voltage step is applied to the Pockel's Cell. Triggering of the
quarter-w ave voltage step is synchronized w ith the tim ing of the Nd:YAG
pum p source. Here, a small portion of the light that pum ps the Ti:Sapphire
cyrstal is used to trigger (activate) the Pockel's Cell. The Pockel's Cells timing
cycle is derived from a fast photodiode w hich m onitors the 76 M H z pulse
train derived from the fem tosecond laser. After an initial delay of less than
13 nanoseconds, a quarter-w ave voltage step is applied w ith 1 nanosecond
variable delay increm ents. Because of the relatively long excited state lifetime
of TirSapphire, the accum ulated delays have a m inim al effect on the seed
pulses ability to extract the energy.1 1
The quarter wave voltage step traps a single pulse inside the am plifier
cavity as follows. First, a selected pulse passes through the Pockel's Cell twice
w hich again leads to a 90 degree polarization rotation (vertical to horizontal).
After the pulse enters the am plifier cavity, an additional voltage step of
several thousand volts is applied to the KDP crystal. This voltage step leads to
an acrhom atic, half-wave birefringence for the KDP crystal. Consequently,
the polarization of the pulse rem ains horizontal after two consecutive passes
through the Pockel's Cell. Thus, as long as the quarter-w ave voltage step is
applied, the horizontally polarized pulse rem ains trapped inside the am plifier
27
cavity. As stated earlier, however, the pum p Nd:YAG laser deposits a finite
am ount of energy to the Ti:Sapphire am plifier crystal. Since there is an
u ppper lim it to the am ount of energy the seed pulse can extract, the am ount
of time the quarter w ave voltage step is applied is set at or near the m axim um
energy acquired from the am plified pulse train.8 H ow ever, there are two
reasons w hy, in som e system s, this m axim um energy point is not used to
couple out the am plified pulse. For example, one group has found that the
shortest pulses are obtained by changing the num ber of round trips in the
am plifier in order to change the net dispersion.8 Thus, to m inim ize net
dispersion arising from m ultiple passes through the am plifier, the pulse is
coupled out before the m axim um pulse energy is reached. O ur group, on the
other hand, found that if you couple out the am plified pulse beyond the
m axim um energy point, the pulse-to-pulse stability im proves dram atically,
although other factors are also involved.11
In order to couple the amplified pulse out of the am plifier cavity, an
additional quarter-w ave voltage step is applied w hereby a round trip through
the Pockel's Cell flips the polarization of the pulse from horizontal to
vertical. W ith the 90 degree polarization flip, the vertically polarized,
am plified pulse will escape the amplifier cavity as it is reflected back tow ard
the isolator assem bly via the TFP.
(D) Pulse Compressor
The last com ponent of our fem tosecond am plifier system is a pulse
com pressor. As illustrated below, the parallel arrangem ent of the diffraction
gratings creates a shorter pathlength for the blue com ponents of the pulse.
28
U nder ideal conditions, the com pressor asssem bly w ould exactly cancel the
red chirp introduced into the pulse by the stretcher. H ow ever, additional
positive chirping occurs during the am plification process, prim arily from the
Ti:Sapphire crystal, but also from other optical com ponents such as the
Pockel’ s Cell and Isolator Assembly. Therefore, w hen the pulse has reached
the com pressor, it has accum ulated additional second and third order
dispersion term s that the com pressor alone cannot com pletely nullify. In
order to com pensate for theses additional phase shifts, a prism pair is also
inserted into the am plifier assem bly.4' 6' 8_9' 13
Figure 2.3 Compressor Design used in CPA
(E) Phase Compensator
The prism pair serves the sam e function as that for the laser cavity.
N am ely, it com pensates for any positive chirping introduced by the pulse as it
transverses optical m edia such as lenses and the Ti:Sapphire crystal. Every
29
fem tosecond am plifier system that produces near-transform lim ited pulses
incorporates a prism pair set* although the positioning w ithin the am plifier
cavity is som ew hat arbitrary. Lack of a prism pair set in our am plifier is one
of the prim ary reasons w hy w e are unable to produce transform -lim ited 60
fem tosecond pulses. Typically, our pulse durations are around 120
fem toseconds. A nother factor preventing us from achieving transform -
lim ited pulses is our use of a lens, and not a spherical m irror, in the stretcher
assem bly. Because of its reflective and not refractive focusing properties, a
spherical m irror avoids the tem poral broadening caused by a pulse
transm itting through optical m aterials, such as a lens. Consequently, excess
dispersion created by the stretcher assem bly is avoided.4-8 Figure ... show s
o u r current o u tp u t param eters.
Femtosecond Laser Parameters
Pulse-to-Pulsc
Energy Fluctuations
0.42%
Repetition Rate
20 Hz
Pulse Duration
120 fs FWHM
0 1
>
< u
E
g o
V
c
tu
Figure 2.4 Current Output from our Femtosecond Amplifier
30
2.4 References
1. H.C. K apteyn and M.M. M urnane, O ptics and Photonics N ew s. M arch
1994,20-28
2. W.L. W eaver adn T.L. G ustafson, Spectrochim ica Acta Rev.. 1993, 15, 527-
579
3. W .H. Knox, IEEE Tournal of Q uantum Electronics. February 1988, 24, 388-
397
4. J. Z hou, C.P. H uang, H.C. K apteyn, and M.M. M urnane, Ultra fast
Phenom ena IX. 1994, 56, 327-329
5. R.L. Fork, C.V. Shank, C. H irlim ann, R.Yen, and W.J. T om linson, O ptics
Letters. A ugust 1, 1983,1-3
6. J. Zhou, C.P. H uang, C. Shi, M.M. M urnane, and H.C. K apteyn, Optics
Letters. January 15,1994,19,126-128
7. M.K. Reed, M.S. A rm as, M.K. Steiner-Shepard, and D.K. N egus, Optics
Letters. M arch 15,1995, 20, 605-607
8. K. W ynne, G.D. Reid, and R.M. H ochstrasser, O ptics Letters. Tune 15,1994,
19,895-897
9. T. Joo, Y. Jia, and G.R. Fleming, Optics Letters, February 15, 1994, 20,389-391
10. S.Backus, J. Peatross, C.P. H uang, M.M. M urnane, and H.C. K apteyn,
Optics Letters. October 1,1995,20,2000-2002
11. F.P. Strohkendl, D.J. Files, and L.R. Dalton, T . Opt. Soc. Am. B,. M ay 1994,
742-749
12. M.D. Perry and G. M ourou, Science. May 13,1994, 264, 917-924
13. C.P.J. Barty, B.E. Lemoff, and C.L. G ordon III, U ltrafast Phenom ena IX.
1994,56,327-329
14. J. Squier, G. Korn, G. M ourou, G. W aillancourt, and M. Bouvier, O ptics
Letters. April 15,1993,18, 625-627
15. O perator's M anual for C oherent M ira M odel 900 Laser
31
16. W.E. W hite/ F.G. Patterson, R.L. Combs, D.F. Price, and R.L. Shephard,
Optics Letters. A ugust 15,1993,12
32
Chapter 3
Femtosecond DFWM of the Photosynthetic Bacteria Rhodobacter
Sphaeroides
3.1 Introduction
A dvances in fem tosecond lasers and am plifiers has led to several
opportunities to stu d y ultrafast phenom ena in various form s of m atter.1
U ntil recently, the prim ary focus of fem tosecond spectroscopy w as the study
of ultrafast electron dynam ics in sam ples as diverse as sem iconductors,
nonlinear organic optical m aterials, inorganic crystals, etc.1 H ow ever,
fem tosecond spectroscopy has also been used to study ultrafast nuclear
dynam ics as dem onstrated repeatedly during the past tw o years.2
M any system s investigated by ultrafast laser techniques have displayed
oscillatory behavior.3' 6 For the m ost part, the oscillations have been
attributed to vibrational coherences in w hich the ultrashort pulse creates a
non-stationary vibrational w avepacket that persists several fem toseconds to
picoseconds after the pulse has passed. W hile the num ber of system s
exhibiting oscillatory features continues to increase, the specific m odes
responsible for the oscillatory behavior and their relevance rem ains unclear.
Perhaps the m ost studied system that displays pronounced oscillatory
features is the photosynthetic reaction center in bacteria.7"11 In this exam ple,
ultrafast laser techniques have been used to stu d y the initial event in bacterial
p h o to sy n th esis12"13 w hich is the 17 angstrom through space electron transfer
from a bacteriochlorophyll dim er to a bacteriopheophytin acceptor. This m ost
33
fundam ental of chemical reactions occurs on a subpicosecond timescale,
extrem ely fast considering the distance betw een the donor and the acceptor
m olecules.6' 14
3.2 Oscillatory Features of Photosynthetic Reaction Centers of
Bacteria
To date, femtosecond spectroscopy of photosynthetic bacteria has largely
focused on studying the effect of amino acid substitutions, via site-directed
m utagenesis, on the initial elecron transfer rate and directionality.15 Yet,
w ith the observation of oscillations in the fem tosecond spectroscopy of the
reaction centers, interest has also focused as to w hether these vibrational
m odes that strongly couple to the P-P* transition m ay assist w ith the extrem e
speed and efficiency of the initial process in photosynthesis. Coherent
electron in the Photosynthetic Reaction Centers has been speculated7 and is
supported by the fact that the oscillations persist during the timescale as the
initial electron transfer process.1 1 If vibrational coherence assists w ith the
electron transfer process, the curve crossing region betw een P* and the
acceptor m ay be reached in a concerted fashion resulting in a macroscopic
electron transfer rate that proceeds in an oscillatory m anner in tim e.10
H ow ever, the vibrational m odes that contribute to the oscillations have yet to
be identified,16 and their relevance to ultrafast electron transfer reaction is
still being debated.8
34
3.3 Bacteriochlorophyll doming as a component in Vibrational
Coherence
O ne possible vibrational m ode that m ay contribute to the oscillations
observed in the photosynthetic reaction center is the M g-dom ing of the
bacteriochlorophyll dim er. This vibrational m ode is characterized by the M g
atom m oving into and out of the plane of the bacteriochlorophyll molecule.
Evidence to support this hypothesis exists, for exam ple, in the FTIR and
R esonance Ram an spectra of isolated chlorophyll m olecules3 w here the
energy (w avenum ber) of the M g dom ing m ode closely resem bles one of the
observed oscillatory com ponents.7* 10
F urther evidence suggests that the M g-dom ing m ay be one of the
oscillatory com ponents in photosynthetic reaction centers of bacteria. For
exam ple, the pum p-probe spectroscopy of the hem e group in m yoglobin, the
oxygen storage protein, show s sim iliar oscillatory w hen excited w th a
fem tosecond laser pulse that is resonant w ith the Soret band.5' 17-18 In this
exam ple, the ultrashort laser pulse dissociates N O from the hem e complex.
Prior to photoexcitation, the N O m olecule occupies the 6th coordination site
(axial position) of the iron hem e group. The other axial coordination site is
occupied by a histidine residue of the protein. W hen ligated w ith N O , the
iron atom rem ains in the plane of the heme. H ow ever, w hen N O is absent,
the iron atom is only coordinated w ith five ligands, and thus rests slightly out
of the hem e plane in a puckered configuration. A fter a fem tosecond pum p
pulse resonantly excites the N O bound hem e group, the N O m olecule
dissociates w ith a tim e constant <60 fem toseconds. W ithin a few picoseconds,
the Fe atom begins to oscillate into and o u t of the plane of the hem e, causing
35
a dam ped sinusoidal profile in the pum p-probe spectroscopy of this
com plex.17-18
In sim iliar fashion w ith the iron hem e group in m yoglobin, the
bacteriochlorophyll dim er in the photosynthetic reaction center likew ise has a
histidine residue that ligates the Mg atom at one of the axial sites.19 H ow ever,
the sixth site rem ains unoccupied d u e to the bacteriochlorophyll dim er or
sandw ich.
3.4 Rational for studying the PRC with Femtosecond DFWM
The goal of studying the photosynthetic reaction center w as m anyfold.
First, I w anted to verify if DFWM w ith fem tosecond pulses w as sensitive to
oscillations in photosynthetic reaction centers of bacteria. Oscillations w ere
previously observed by our group from DFWM m easurem ents of
vibrationally restricted ladder-type chrom ophores. M ore recently, N elson at
MIT has found that fem tosecond DFWM is sensitive to nuclear coherences in
different phases of m atter. O ther ultrafast optical techniques that have also
been show n to be sensitive to oscillatory behavior include pum p-probe,
fluorescence up-conversion, and the Optical Kerr Effect.2
A nother reason w hy I w anted to exam ine the photosynthetic reaction
center w as that w e had earlier problem s w ith m easuring DFWM signals from
sam ples that did not have significant absorption w ithin the fundam ental
tuning range of our TkSapphire am plifier system . As stated previously, our
current tuning range is betw een 740 to 880 nm , ideal for studying the
photosynthetic reaction center, particularly R hodobacter Sphaeroides w hich
has m easurable absorption from 700 to 900 nm .20
36
m -no t - o m ~ m «o
Figure 3.1 Visible Spectrum of Rhodobacter Sphaeidores
W ith o u r w avelength agility, w e could also com pare electron transfer kinetics
w ith the P-P* transition from 840-880 nm.
W hat follows next is our first experim ental results from fem tosecond
DFWM of the photosynthetic reaction center from the w ild-type version of
R hodobacter Sphaeroides.
3.5 Experimental Protocol, Measurements, and Data
W ild type R hodobacter Sphaeroides reaction centers w ere kindly
provided by Dr. D eborah H ansen from A rgonne N ational Laboratory. The
reaction centers w ere contained in the follow ing buffer solution:
10 m M Tris-CI, PH 8.0
0.09% LDAO (V /V )
0.1 mM EDTA
1% sodium azide (W /V )
The sam ple w as stored at -20 degrees celsius until m easurem ents could be
taken. O nce the laser w as available, the photosynthetic reaction center w as
thaw ed to room tem perature and placed in a 1 m m optical cell. N ext, the UV-
Vis spectra was taken from 300 to 900 nm in order to verify that the protein
had not degraded since delivery. To check the stability of the protein
complex, the relative absroption peak heights at 760, 800 and 860 nm w ere
com pared. Ratios of 1:2:1 are expected. If degradation is present, the 860 nm
peak will get sm aller and the 760 nm peak will increase.19 C om parison of the
peaks revealed no degradation prior to the experim ental m easurem ents. A t
the tim e the fem tosecond DFWM m easurem ents w ere taken, the laser was
tuned at 776 nm. The optical density at this w avelength w as approxim ately
0.2, and the laser pulses were 120 femtoseconds.
Next, the data we generated will be presented and reviewed. W hile
oscillations were not evident, the signal w e obtained was one of the best we
have encountered, and very com parable to recently published results.21
M oreover, interesting data recently reported from fem tosecond DFWM
m easurem ents of isolated bacteriochlorophyll molecules in solution22 may
also suggest additional experim ents that w e could try in order to detect
oscillations by our experim ental set up.
Figure show s our first encounter w ith the photosynthetic reaction center.
W hile oscillations appeared initially, subsequent m easurem ents.
•2 0 0 0 -1 0 0 0 0 1000 2 0 0 0 3 0 0 0 4 0 0 0
____________________Beam D elay (femtoseconds)___________________
Figure 3.2 Initial pre-filtered Femtosecond DFWM Signal
38
show ed no obvious oscillatory features. In order to check for possible
photodegradation, I ran another UV-Vis spectrum . H ow ever, no apparent
change in the relative peak heights w ere observed. After a few m ore
experiental runs, the sam ple was filtered w ith 0.2 m icron pore size syringe.
Subsequent m eausrem ents gave very clean signals w ith no unusual features.
A pparently the first curve we generated and others prior to filtration were
contam inated w ith dust or particulate m atter. The sensitivity of the DFWM
signal to dust or small particles necessitates that the sam ples be filtered prior
to m easurm ent.
Q 0 . 4 -
1500 2000 5 00 1000
Beam Delay (fem toseconds)
Figure 3,3 Post-filtered Femtosecond DFWM Signal
As indicated from the post-filtered curve, no oscillations are readily
apparent.
W hile oscillations are not evident from our DFWM m easurem ents,
others have reported them w ith the sam e sam ple. Is there an explanation?
Fortunately, there are several. First and foremost, the w avelength w e had
available for experim entation, 776 nm , is consistent w ith excitation of the
39
accessory bacteriochlorophylls of the photosynthetic reaction center.20* 21 As
reported recently,2 1 w ithin 100 fem toseconds after photoexcitation, the
accessory bacteriochlorophylls transfer their energy to the special pair which is
subsequently used for ultrafast electron transfer. Oscillations w ere reported
for excitation of Rhodobacter Spharoides w ith 27 fem tosecond pulses
centered at 803 nm.
Clearly, any possibility of view ing oscillatory features in the
photosynthetic reaction center requires that the P-P* transition be excited. As
has been reported previously,9* 1 1 the oscillations are associated w ith P \
W hile w e have the available tuning to excite a portion of this region, it is not
readily available for experim entation at this time. Even if our operating
w avelength is tuned w ithin the 840 to 880 nm region, several factors m ay
prevent us from observing oscillatory features in our signal. For exam ple, the
w ild type version of Rhodobacter Sphaeroides has the least persistent
reported oscillations.9 Furtherm ore, the oscillations are m ost apparent w hen
w orking at cryogenic tem peratures.10 Currently, all of o u r experim ents are
perform ed at room tem perature. Yet, recent reports do indicate that
oscillations are still discernible, even at room tem perature and for several
different syterns.3* 4' 9
Perhaps the strongest factor that m ay prevent us from observing
oscillations is the lack of transform -lim ited pulses produced from our
am plifier system. Pulse durations of 100 fem toseconds or m ore have been
reported to be less sensitive to oscillations than w ith pulses of 60
fem toseconds or less. In particular, the use of relatively long pum p and probe
pulses have been show n to sm ear our oscillatory features.9 A lthough our
pulses have enough frequency content to produce 60 fem tosecond pulses, our
40
experim entally derived pulse duration is only 120 fem toseconds. Beyond any
doubt, the major obstacles preventing us from achieving transform lim ited
pulses is our stretcher assembly which uses refractive and not reflective
optics, and our lack of a prism pair in our am plifier cavity. A t this time it is
not yet evident w hether the relatively sim ple rem edy for this problem will be
pursued or not.
Because of our relatively large tuning range, it w ould be interesting to
perform w avelength-dependent m easurem ents from 840-880 nm and
com pare the kinetics of the initial charge transfer reaction. Besides
potentially observing the desired oscillatory features, this experim ent could be
particularly relevant to the coherent nuclear control of electron transfer rates.
If electron transfer occurs in an oscillatory m anner, as has been suggested,10
w avelength-dependent kinetics should be observed. A nother recently
reported result from fem tosecond DFWM of isolated bacteriochlorophyll
molecules suggest even further experim ents in w hich changing the
polarization of the probe beam m ay be sensitive to vibrational m odes norm al
to the bacteriochlorophyll plane.22 This result may also be relevant to the
photosynthetic reaction center. Surprisingly, and w ith great relief, w e saw a
sim iliar signal from isolated chlorophyll molecules, the results of w hich will
be described next.
3.6 References
1. See, for example, the series on U ltrafast Phenom ena
2. L. Dhar, J.A. Rogers, and K.A. Nelson, Chem. Rev.. 1994, 94, 157-193
41
3. M. Chachisvilis, H. Fidder, T. Pullerits, and V. Sundstrom , Tournal of
Raman Spectroscopy. 1993, 26, 513-522
4. E. Lenderink, K. D upper, and D.A. W iersma, T . Phvs, Chem, 1995, 99, 8972-
8977
5. L. Zhu, P. Li, M. H uang, J.T. Sage, and P.M. Cham pion, Phvs. Rev. Let.,
January 10,1994, 72, 301-304
6. Q. W ang, R.W. Schoenlein, L.A. Peteanu, R.A. M athies, and C.V. Shank,
Science. October 21,1994,266,422-424
7. M.H. Vos, F. Rappapart, J.C. Lambry, J. Breton, and J.L. M artin, N ature,
May 27,1993, 363,320-325
8. R.J. Stanley and S.G. Boxer, T . Phvs. Chem. 1995, 99, 859-863
9. M.H. Vos, M.R. Jones, C.N. H unter, J. Breton, and J.L. M artin, Proc.
Natl.Acad. Sci. USA, December 1994, 91, 12701-12705
10. M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.C. Lambry, and J.L. M artin,
Biochemistry, 1994, 33, 6750-6757
11. M.H. Vos, J.C. Lambry, S.J. Robles, D.C. Youvan, J. Breton, and J.L. M artin,
Proc. Natl. Acad. Sci. USA. October 1991, 88,8885-8889
12. G.R. Fleming and R.V. Grondellle, Physics Today. February 1994, 48-55
13. G.R. Fleming, J.L. M artin, and J. Breton, N ature, M ay 12, 1988, 333, 190-192
14. J.R. N orris and M. Schiffer, C&EN. July 30,1990, 22-37
15. J.W. Jia, T.J. Dim agno, C.K. Chan, Z.Y. Wang, M. Du, D.K. Hanson, M.
Schiffer, J.R. Norris, and G.R. Fleming, T . Phvs. C hem .. 1993, 97, 13180-
13191
16. S. Maiti, G.C. W alker, B.D. Cowen, R.Pippenger, C.C. Moser, P.L. Dutton,
and R.M. Hochstrasser, Proc. Natl. Acad. Sci. USA. October 1994, 91, 10360-
10364
17. L. Zhu, J.T. Sage, and P.M. Champion, Science. October 28,1994, 266, 629-
632
42
18. L. Z hu, W. W ang, J.T. Sage, and P.M. C ham pion, Tournal of Ram an
Spectroscopy. 1995,26,527-534
19. Personal correspondence w ith Dr. Debbie H anson, A rgonne N ational
Laboratory
20. W. Z inth and W. Kaiser, The Photosvnthetic Reaction Center: Volum e II,
1993,71-88
21. Y. Jia, D.M. Jonas, T. Joo, Y. N agasaw a, M.J. Lange, and G.R. Fleming,
T . Phvs. C hem .. 1995,17, 6263-6266
22. G.P. W iederrecht, W.A. Svec, M.P. N iem czyk, and M.R. W asielew ski, L_
Phvs. C hem .. 1995, 99, 8919-8926
43
Chapter 4
Femtosecond DFWM of Metal-Substituted Chlorophyll-a and
Bacteriochlorophyll-a
4.1 Introduction
D uring the time I was investigating the fem tosecond spectroscopy of the
bacterial reaction centers, I was also developing two separate projects which
were also designed to study possible ultrafast vibrational coherences. The first
project involved isolation and chemical m odifications of bacteriochlorophyll
and chlorophyll molecules, w hile the second pertained to a m onoclonal
antibody that binds to a chrom ophore which undergoes a trans to cis
photoisom erization. Since the form er project w as m ore directly related to
vibrational coherences observed in photosynthetic reaction centers of bacteria,
details of this project will be given next. Thereafter, the latter project, which
m odels the other photosynthetic bacteria bacteriorhodopsin, will be review ed
in the closing chapter.
4.2 Experiemental Rational for Metal-Substituted Chlorophyll-a
As described in the previous chapter, several published results supports
the concept of ultrafast hem e dom ing in m yoglobin and hem oglobin.1
Consequently, it is likely that sim iliar dom ing m odes of bacteriochlorophyll
appear as oscillations in the ultrafast spectrocopic studies of photosynthetic
reaction centers.2 Because of my particular interest and focus in the m etal
44
(m agnesium ) center of the bacteriochlorophyll molecule, I decided to
substitute heavier divalent m etal ions in the bacteriochlorophyll m olecule
w ith the hopes that vibrational coherence w ould be observed, and that such
oscillations w ould have different periods.
Bacteriochlorophyll was the ideal candidate to perform the m etal
substitutions for the following reasons. First, the oscillations are observed in
photosynthetic bacteria w hich contain bacteriochlorophyll m olecules. The
diference betw een bacteriochlorophyll and chlorophyll is given in the
follow ing figure.
.o
CH3
CH.
C hlorophyll-a
B acteriochlorophyll-a
R =
o
Figure 4.1 Chemical Structures of Chlorophyll-a and
Bacteriochlorophyll-a
45
Likewise, bacteriochlorophyll-a has overlapping absorption spectrum w ith
o u r available tuning range as show n in figure.
Figure 4.2 Absorption Spectrum of Bacteriochlorophyll-a
in Acetone
W ith these tw o factors, along w ith our earlier and continuing problem s w ith
sam ples that do not have significant absorption w ithin the available tuning
range, bacteriochlorophyll-a appeared to be the ideal m olecular m odel for
vibrational coherence studies. Unfortunately, a few factors prevented me, at
the time, from perform ing metal substitutions on bacteriochlorophyll-a
molecules. First, the cost of bacteriochlorophyll-a was very prohibitive, w ith
the least expensive being $35.00 per milligram. Also, until recently,3 no
literature preparation was given for m etal substitutions of
bacteriochlorophyll-a molecules. On the other hand, several literature
preparations w ere available for metal substitutions of the related chlorophyll-
a m olecule.4* 6 M oreover, chlorophyll-a m olecules, w hile as expensive as
bacteriochlorophyll-a molecules, can be easily isolated and purified by
standard chemical techniques.7'8 This is in contrast w ith bacteriochlorophyll-
a w hose isolation and purification requires more elaborate chemical
instrum entation (HPLC).9 In addition, I found chlorophyll-a had superior
chemical stability com pared to its bacteriochlorophyll counterpart.
46
U nfortunately, a m ajor draw back of chlorophyll-a w as that its relevant Qy
absorption band resides around 660 nm , beyond o u r available tuning w indow
of 740 nm to 880 nm. Therefore, to p u rsu e fem tosecond DFW M experim ents
on chlorophyll-a and m etal substituted chlorophyll-a m olecules, tw o criteria
m ust be satisfied. First, the laser m ust be tuned to the blue end (740 nm ) of
our tuning range. Also, the optical density at this w avelength should roughly
be 0.2, as w e learned from our previous experience w ith Professor H offm an's
phthalocyanine sam ples. To achieve the necessary optical density, the sam ple
w ould have to be higly concentrated w hich m eant the isolation and
purification of several h u n d red m illigram s of chlorophyll-a m olecules. This
task w as undertaken for a few m onths, and the procedure used as well as the
results obtained from fem tosecond DFWM at 740 nm will be described next.
4.3 Isolation and Purification of Chlorophyll-a from Spinach
As m entionned earlier, several references are available for both the
isolation and purification of chlorophyll-a m olecules as well as the respective
m etal substitution reactions. To start, I choose to isolate an d purify
chlorophyll-a m olecules from a procedure w hich used three readily available
solvents (acetone, w ater, and isooctane) as well as tw o colum n
chrom atographic packing m aterials (polyethylene and pow dered sugar).8
A long the w ay, I perform ed a few m odifications, as needed, to satisfactorily
isolate an d purify chlorophyll-a m olecules. The procedure called for 200
gram s of fresh spinach that w as subsequently w ashed and dried. The
pigm ents contained in the spinach extract include the chlorophylls, the
xanthophylls, the pheophytins, and the carotenes. To extract these
47
com ponents, the 200 gram s of fresh spinach leaves w ere im m ersed in 500 ml
of reagent grade acetone. To separate the pigm ents and isolate the
chlorophylls, the extract w as diluted w ith w ater to give an 80% w ater-acetone
solution. The aqueous extract w as applied to a 5 cm glass colum n packed w ith
m edium density polyethylene w hich had been w ashed w ith 70% aqueous
acetone. Actual application of the extract to the packed colum n required two
m odifications from the referenced procedure. First, the polyethylene was
applied as a slurry instead of dry packing as indicated in the procedure.
Second, and likely a consequence of the first m odification, only about half of
the 500 ml of extract w as applied to the colum n. O therw ise, the colum n
w ould becom e saturated w ith extract, leaving an incom plete separation of
the com ponents. Once applied to the colum n, the pigm ents separated as
follows: xanthophylls, chlorophylls, and carotenes. A ccording to the
procedure, the pheophytins should also have appeared in betw een the
chlorophylls and the carotenes. H ow ever, after num erous colum n runs, no
sign of the brow nish pigm ent w as ever observed.
A nother set of m odifications w as w ith regards to the eluting solvent.
First, the extract w as diluted w ith w ater to give a 80% w ater-acetone solution.
H ow ever, on several occasions, the xanthophylls and chlorophylls w ould not
com pletely separate, w hich w ould becom e evident w hen the next step, a
solvent exchange reaction, w as perform ed. To give better separation of these
tw o com ponents on the colum n, I w ould typically dilute the extract even
further to give a 70% to 75% aqueous acetone extract. W hen this was
perform ed, separation of the xanthophylls from the chlorophylls becam e
com plete. The procedure also required that the chlorophylls w ould be m ore
easily eluted from the colum n if an 85% aqueous acetone solution w as used.
48
H ow ever, I found that the 70% aqueous acetone solution, w hich w as used to
com pletely elute the xanthophylls, w orked just as w ell in eluting the
chlorophylls. Thus, the 85% aqueous acetone solution w as only used
occasionally.
A fter the chlorophylls w ere separated and collected, the next step w as to
exchange the solvent from polar to nonpolar. The nonpolar solvent used
w as isooctane. The solvent exchange w as carried o u t as follows.
A pproxim ately 500 ml of isooctane and an equal am ount of chlorophylls in
aqueous acetone w ere added to a separatory funnel. Because of occasional
incom plete separation of the xanthophylls from the chlorophylls
(m entionned above), the low er aqueous acetone layer w as som etim es yellow.
W hen the yellow color appeared, I w ould rinse the organic (chlorophyll)
fraction w ith 3 to 5 equal volum e portions of 70% aqueous acetone solution.
Tow ard the latter p art of m y chlorophyll isolations, w hen I used a m ore
dilute extract solution, additional aqueous acetone rinsing of the chlorophyll
fraction w as not necessary since the colum n w ould com pletely separate the
xanthophylls from the chlorophylls.
For the m ost part, separation of the chlorophylls follow ed as indicated by
the procedure w ith only one m inor m odification. Once the chlorophylls
w ere transferred into the isooctane solvent, the next step was to apply this
solution (approxim ately 200 ml) to a pow dered sugar colum n w hich w as
likew ise prepared by an isooctane slurry. A fter the chlorophyll solution w as
ad d ed to the pow dered sugar colum n, com plete separation of the chlorophylls
(a and b) w as achieved w hen the eluting isooctane solvent w as spiked w ith
0.5% of n-propanol. The bluish-green chlorophyll-a eluted first w ith the
yellow -green chlorophyll-b lagging far behind.
49
A lthough the procedure w orked fairly well, the actual preparation of the
colum n was very tedious. Air gaps w ere a com m on occurrence in the
pow dered sugar colum n, leading to incom plete separation and otherw ise
com plete failure of the entire process. Special care w as essential in order to
avoid the air gaps. Otherwise, the entire process w ould have to be repeated
again.
W hen a suitable pow dered sugar colum n w as obtained, the separation was
tediously slow. To speed up the separation, a flash colum n w as used. Both
techniques w ere tried. Nam ely, pressure was applied to the top of an
approriate fitted colum n or a vacuum line was connected at the extension of
an erlenm eyer flask. For covenience of space, the vacuum m ethod, once
perfected, was used the most. The final separations obtained by the flash
colum n technique w ere excellent and the tim e it took w as reduced
considerably.
U nder ideal circumstances, I w ould have collected the chlorophyll-a
molecules in individual batches. But because of time constraints and the
uncertainty of actually obtaining the desired product, I collected as m uch of
the chlorophyll-a and saved all of it in one batch. The purity of the
chlorophyll-a molecules w ere ascertained by the m ethod used in the literatue
procedure,8
4.4 Metal Substitution reactions of Chlorophyll-a
Final m anipulation of the chlorophyll-a m olecules involved selective
m etal substitution reactions. As with the isolation of chlorophyll-a molecule,
num erous prcedures are available to perform this sim ple exchange reaction.4*
50
6To me, the easiest m ethod was to use the respective acetate salt and mix
them w ith the pheophytin-a complex as suggested by professor Chris Reed
(USC D epartm ent of Chemsitry). The procedure I used for this reaction was
as follows. A pproxim ately 100 ml of the chlorophyll-a solution was allow ed
to evaporate in a fum ehood in order to rem ove the isooctane solvent. W hen
the isooctane w as rem oved, the dark, waxy green chlorophyll-a sam ple was
dissolved w ith acetone. A 0.1 M HC1 solution was also prepared and 5
volum e parts of the chlorophyll-a acetone solution w as added to a three neck
round bottom flask along w ith one volum e part of the 0.1 M HC1 solution.
A lthough not absolutely necessary, I carried this reaction under an inert
argon atm osphere. After a few seconds w ith constant stirring at room
tem perature, the green solution turned brow n, transform ing the chlorophyll-
a m olecule to the respective pheophytin-a m olecule as depicted in the figure
below:
C H . C H .
Acid
CH
C H .
CH CH
CH CHj
C hlorophyll-a Pheophytin-a
Figure 4.3 Acid Addition to Chlorophyll-a to form Pheophytin-a
51
After 10 to 15 m inutes, the pheophytin-a reaction m ixture was transferred to
an equal volum e of isooctane. The pheophytin-a subsequently partitioned to
the organic (isoocatane) layer. After discarding the aqueous acetone reaction
m ixture, the pheophytin-a solution w as w ashed w ith 3 to 5 equal volum e
portions of deionized water. Next, the w ashed pheophytin-a solution was
dried over anhydrous sodium sulfate crystals.
To perform the m etal substituion reaction, the isooctane w as rem oved
from the pheophytin-a solution. Once the waxy, brow n pheophytin-a
com pound was dried, approxim ately 150 ml of glacial acetic acid was added to
m ake the respective pheophytin-a solution. Into a three neck round bottom
flask, the pheophytin solution was added. The metal salt was prepared by
dissolving 1 to 2 gram s in approxim ately 150 ml of glacial acetic acid. After
com plete dissolution, which occasionaly took vigorous shaking, the selected
m etal salt w as added to the reaction m ixture. All m etal substituions reactions
w ere conducted in an inert argon atm osphere w ith constant stirring. The
reaction tem perature was m aintained at roughly 60 degrees Celsius. After
approxim ately 20 m inutes, the reaction m ixture w as cooled to room
tem perature, w hich took 10 to 15 m inutes. The reaction m ixture w as
m aintained under the argon atm osphere during the entire cooling process.
After cooling, the m etal-substituted chlorophylls w ere isolated as follows.
A pproxim ately 100 ml of isooctane was added to the m etal substituted
chloroophyll-a solution. Since no separation occured, an equal am ount of
deionized w ater w as added which subsequently lead to the acetic acid
separating from the isooctane layer to the w ater layer. After discarding the
aqueous layer, the rem aining isooctane solution of m etal substituted
chlorophylls w ere w ashed w ith 3 to 5 equal volum e portions of deionized
52
w ater and dried over anhydrous sodium sulfate crystals. A lthough I could
have stopped here and m ade the fem tosecond DFWM m easurem ents with
the isoocatane solvent, I decided to exchange the solvent w ith acetone. Thus,
I evaporated the isooctane until dried and added the appropriate am ount of
acetone.
CH
C H .
M ✓
M etal Acetate
Reflux
M =Cu, N i, Zn
C H .
C H
C H .
C H .
C H .
CHj
M etal-Subtituted Chlorophyll-a
Figure 4.4 Metal Insertion Reaction
As stated earlier, one of the potential disadvantages of substituting
bactericohlorophyll-a with chlorophyll-a is that it lacks significant absorption
w ithin our available laser tuning range. Thus, in order to observe a signal,
the chlorophyll-a sam ples w ould have to be concentrated to the point w here a
certain optical density at the operating wavelength is achieved. Luckily, at the
tim e the m easurem ents were perform ed, the laser w as tuned at 738 nm.
H ow ever, the chlorophyll-a sam ples still needed to be concentrated.
53
For the metal substitutions I performed, figure shows their resepective
absorption spectrum in acetone as well as the native chlorophyll-a molecule.
i
5 0 0 4 0 0 W O 7 0 0
Figure 4.5 Chlorophyll-a UV-Vis Spectrum
IM 3 0 0 4 0 0 1-00 <:00 71)0 :;iM>
Figure 4.6 Copper Chlorophyll UV-Vis Spectrum
IM 3 0 0 4 0 0 0 0 0 5 0 0 7 0 0 3 0 0
Figure 4.7 Nickel Chlorophyll UV-Vis Spectrum
IM 3 0 0 4 0 0 w o w o 7 0 0 "110
Figure 4.8 Zinc Chlorophyll UV-Vis Spectrum
54
4.5 Femtosecond DFWM Measurements
0 .5
7 5 0 0 5 0 0 0 2 5 0 0
Beam Delay (fs)
Figure 4.9 Femtosecond DFWM Signal of Copper Chlorophyll
S. 0.22
o >
0.20
0.18
0.16
0.14
5 0 0 0 7 5 0 0 2 5 0 0 0
___________________________Delay Beam ffs)_________
Figure 4.10 Enlargement of Figure 4.10
55
For the initial m easurem ents, I prepared the chlorophyll-a sam ples w ith
optical densities of approxim ately 1.0 at the m axim um near infrared
absorption region (around 660 nm). H ow ever, at this optical density no signal
from the chlorophyll-a sam ples w as observed. Therefore, I concentrated the
sam ples further to the point w here the absorption at 738 nm w as roughly 0.3.
W hen the fem tosecond DFWM experim ents w ere perform ed, extrem e
scattering w as observed. To clean the sam ples, I filtered them w ith 0.2 m icron
pore size syringes.
M ost o f the sam ples did not give a signal except the copper substituted
chlorophyll. Surprisingly, an ultrafast com ponent is observed w ithin 500
fem toseconds. At about the sam e tim e these m easurem ents w ere m ade, I
obtained a recently published paper w hich also exam ined the fem tosecond
"transient grating" of chlorophyll m olecules in solution.10 This publication
also reported a sim iliar signal from bacteriochlorophyll-a at 700 nm as show n
in figure below.
fl »-72?
r -5 2 0 fs
*1.00 *0,50 0.00 0.50 1.00 1 .5 0 2 .0 0 2.50
TIME ps
Figure 4.11 transient grating results
56
Two physical processes have been speculated to possibly contribute to this
unusual signal: intram olecular vibrational relaxation, or the form ation of
an ultrafast excited state that involves changes in the bond betw een the
central atom and a ligated solvent m olecule.10 Since this signal w as only
recently published, along w ith our ow n results, m uch m ore w ork will need to
be done in order to understand the actual process w hich is occuring.
It appears from these results that m etal substitutions is one w ay to further
investigate this phenom ena. As stated in the cited article, the signal only
appears in m etalated chlorins and bacteriochlorophyll-a w hen they are probed
w ithin the Qy(l,0) band.10 Due to other obligations w ith the laser, nam ely the
incorporation of an Optical Param etric Am plifier, as well as the lack of
available tuning to lower the already saturated solution concentration, I was
unable to perform further m easurem ents. O bviously, there are several
directions to take w ith this project if additional tuning regions become
available w ith our laser system.
Two m ore results also give further credence and optim ism to this project.
First, o u r previous experience w ith professor H offam ann's phthalocyanine
sam ples led us to speculate that in order to observe a fem tosecond DFWM
signal, the closer you are to a resonant peak, the better. H ow ever, of the six
sam ples only a few had absorption close to the outer limits of our available
tuning. N one of the sam ples had any significant absorption at 767 nm , which
w as w here w e w ere operating w hen we first m easured the sam ples. Since w e
w ere now operating at 738 nm , I decided to to look at one of the Hoffm ann
sam ples again. The sam ple was placed in acetone, filtered, and concentrated
to an optical density of 0.3. The following figures show s the com pound, its
spectra, and resulting Fem tosecond DFWM Signal.
57
Figure 4.12 Phthalocyanine sample examined by Femtosecond
DFWM at 738 nm
<=!.».009 n
H H I - 0 . O O M r t
33.9 ’ 290.0 990.0 «0.0 590.0 634,0 730.0
Figure 4.13 UV-Vis Spectrum of Phthalocyanine Sample
58
*3 0 .2 0 -
0 .10-
0 . 0 5 -
5 0 0 0
Beam D elay (fs)
7 5 0 0 2 5 0 0
Figure 4.14 Femtosecond DFWM Signal from Pthalocyanine
W ithout a doubt, the sim ple m anuever of tuning the laser closer to an
absorption peak peak greatly aided in observing a fem tosecond DFWM signal.
Because saturation was preventing us from observing the decay dynam ics, I
diluted the signal and got the surprising results show n in the accom panying
figures. Clearly, our previous result with copper chlorophyll was not an
artifact, as successive experim ental runs gave reproducible signals.
O ther encouraging new s was that incorporation of m etal substituted
bacteriochlorophyll-a m olecules has recently been accom polished w ith a full
procedure now in press.3 W ith our current tuning range, along w ith a
procedure to produce m etal substituted bacteriochlorophyll-a molecules, the
essence of the signal could be readily investigated.
59
4.6 Vibrational Coherence in isolated Chlorophyll Molecules?
A lthough this was not the signal I had anticipated, are there still
observable oscillations in isolated chlorophyll or bacteriochlorophyll
molecules? An earlier publication w ith fem tosecond pum p-probe data of
bacteriochlorophyll-a m olecules in solution at room tem perature stated they
observed no oscillations.11 However, a recent fem tosecond pum p-probe
investigation of bacteriochlorophyll-a at 4.2 kelvin yielded a reproducible
oscillation w ith a vibrational w avenum ber of 104 cm*1, very sim ilar to one of
the oscillatory com ponents observed in Rhodobacter Sphaeroides. 12 In the
not too distant future, w e too m ay have the capability to perform
fem tosecond DFWM experim ents at cryogenic tem peratures. Despite this,
oscillations are now being reported for other light-activated and non light-
activated biological sam ples at room tem perature.13 My final project, which
w as never com pleted, mimicks the system in which room tem perature
oscillations are observed. I begin my final chapter with som e background on
this project.
4.7 References
1. L. Zhu, W. W ang, J.T. Sage, and P.M. Cham pion, Tournal of Raman
Spectroscopy. 1995,26,527-534
2. See C hapter 3, reference #'s 7-11 and 13.
3. G. Hartw ich, C.Geskes, H. Scheer, J. Heinze, and W. M antele, In Press
4. L.J. Bouch and J.J. Katz, T . Am. Chem. Soc.. 1967, 89,4703-4708
5. R.L. Heald and T.M. Cotton, T . Phvs. Chem.. 1990, 94, 3968-3975
60
6. M. Fujiwara and M. Tasumi, T . Phvs. C hem .. 1986, 90, 5646-5650
7. M. Fujiwara and M. Tasumi, T . Phvs. Chem ., 1986, 90, 250-255
8. A.F.H. A nderson and M. Calvin, N ature. April 26, 1962, 285-286
9. W. Svec, Chlorophylls. H. Scheer Ed., CRC Press, Boca Raton, FI, 1991,89-
102
10. See C hapter 3, reference #3
11. S. Sarikhin and W.S. Struve, Biophysical Tournal. 1994, 67, 2002-2010,
12. See C hapter 3, reference # 3
13. See C hapter 3, reference # 6
61
Chapter 5
Light Acivated Catalytic Antibodies and Bacteriorhodopsin
5.1 Bacteriorhodopsin Background
A nother photosynthetic bacteria w e investigated w ith fem tosecond
DFWM w as bacteriorhodopsin. Unlike the photosynthetic reaction center of
R hodobacter Sphaeioides, bacteriorhodopsin contains only one chrom ophore,
retinal, w hich is bound to the protein via a Schiff base linkage.1
Figure 5.1 Binding Pocket Schematic of Bacteriorhodopsin
The initial ultrafast event in bacteriorhodopsin's extensive photocycle is the
trans to cis photoisom erization along the retinal's C13-C14 bond.2 The
function of the photoisom erization event is to translocate a proton from the
extracellular to intracellular com partm ents of the cell, thus creating an
electrostatic potential difference.3 Subsequently, m em brane-bound proteins
isom erization
bond
isom erization
Asp 212
Asp 85
Arg 82
62
channel the protons back to potentiostatic equilibrium w hile concom ittantly
useing the potential difference to generate the cell’ s energy m olecule, ATP.2
The chrom ophore used by bacteriorhodopsin is nearly identical to the
visual pigm ent found in higher anim als.4 The only difference is that the
retinal chrom ophore is in the cis state along the bond's 12-13 linkage.
Follow ing photoexcitation, rhodopsin undergoes an ultrafast cis to trans
pho to iso m erizatio n .4 H ere, the structural change provides a stim ulus for the
photoreceptor.6
Initially, I began m y research project trying to exam ine the initial and
subsequent ultrafast states of bacteriorhodopsin.2/ 7_10 The extensive electron
delocalization exhibited by the retinal chrom ophore, along w ith its unique
linkage in the protein pocket of bacteriorhodopsin, could potentially lead to
som e interesting nonlinear properties not displayed by the isolated
chrom ophore in solution. Previous nonlinear stu d ies (DFWM) w ere
perform ed w ith nanosecond pulses.11* 12 Recently, hyperayleigh scattering
m easurem ents of bacteriorhodopsin have indicated large m olecular
hyperpolarizability of the protein bou n d retinal chrom ophore.13
H ow ever, the absorption region for photoisom erization w as beyond our
tuning range. This was o u r first experience w ith the tu n ab ility /ab so rp tio n
problem . The signal w e generated w as from the polym er m atrix an d not
photoexcited bR. Several interesting fem tosecond m easurem ents have been
m ade on both bacteriorhodopsin2 and rhodopsin.5 Both have displayed
oscillatory features, w ith the latter's oscillations believed to be funtionally
relevant.5 O scillations have likew ise been observed for ethylene derivatives
in solution w hich have been used to m im ic the photoisom erization reaction
in rh odopsin.14
63
5.2 Photoactive Catalytic Antibodies
D uring the past year, I also developed a m odel visual receptor system that
encom passes two areas of interest. First, I obtained a m ouse m onoclonal
antibody w hich binds specifically to the 2,4-dinitrophenoI group. Through an
azo coupling reaction, the following w ater-soluble chrom ophore was
prepared.
The goal of this project w as to exam ine the dynam ics of the w ell-know n
photoisom erization reaction of azobenzene goups w hen it is bound by the
antibody protein. In fact, during the design period of this project, picosecond
antibody18 was reported as well as that for spiroyans.16' 17
Because of the restricted degrees of freedom im posed on the chrom ophore
by the antibody binding pocket, I have speculated that photoisom erization
rates m ay increase and vibrational coherences, if present, m ay be m ore
pronounced. A nother reason w hy I w anted to use m onoclonal antibodies
was the intense grow th of catalytic antibodies.18 M ore recently, light has been
used of a reagent for catalysis,19 and w ith nuclear rearrangem ents of a
no2
NaO
Figure 5.3 Water-Soluble Hapten Chromophore
for MOPC 315
studies of azobenzene chrom ophores bound inside the pocket of an
64
photoisom erization reaction, I thought this w as an excellent m odel system to
explore potential light induced catalytic effects. H ow ever the absorption
profile of the designed chrom ophore is beyond the fundam ental, b u t
overlapping the second harm onic region of o u r laser.
W ith successful im plem entation of an OP A, w hich could extend o u r tuning
range from 440 nm to 700 nm , both bR, the antibody-bound chrom ophore,
an d the m etal-substituted chlorophylls could be p ursued w ith further
m easurem ents, and hopefully provide m ore insight into these interesting
biological system s.
5.3 References
1. R.A. M athies, S.W. Lin, J.B. Ames, and W.T. Pollard, A nnu. Rev. Biophvs.
Biophvs. Chem .. 1991, 20, 491-518
2. L. Song, M.A. El-Sayed, and J.K. Lanyi, Science. A ugust 13,1993, 261, 891-
894
3. D. O esterhelt, C. Brauchle, and N. H am pp, Q uarterly Reviews of
Biophysics. 1991, 24,425-478
4. L.A., Peteanu, R.W. Schoenlein, Q. W ang, R.A. M athies, and C.V. Shank,
Proc. Natl. Acad. Sci. USA. Decem ber 1993, 90,11762-11766
5. Q. W ang, R.W. Schoenlein, L.A. Peteaun, R.A. M athies, and C.V. Shank,
Science. O ctober 21,1994, 266,422-424
6. Z.T. Farahbakhsh, K. H ideg, and W.L. H ubbell, Science. N ovem ber 267,
1993, 262,1416-1419
7. M.A. El-Sayed, Acc. Chem . Res.. 1992, 25, 279-286
8. R.R. Birge, Scientific A m erican. M arch 1995, 90-95
9. Z. Chen and R.R. Birge, TIBTECH. July 1993,11, 292-300
65
10. R.R. Birge, C om puter, N ovem ber 1992, 56-67
U.S. W u, C. Brauchle, and M.A. El-Sayed, A dvanced M aterials, 1993, 5, 838-
841
12. G.R. K um ar, S.J. W ategaonkar, and M. Roy, O ptics C om m unications, 1993,
98,127-131
13. K. Clays, E. Hendrickx, M, Triest, T. Verbiest, A. Persoons, C. D ehu, and J.L.
Bredas, Science, N ovem ber 26,1993, 262, 1419-1321
14. E. Lenderink, K. D upper, and D.A. W iersm a, T . Phvs. Chem ., 1995,99, 8972-
8977
15. M. H arada, M. Sisido, J. Hirose, and M. N akanishi, Bull. Chem. Soc. Tpn„
1994,67,1380-1385
1 6 .1. W illner, R. Blonder, and A. Dagan, T . Am. Chem. Soc., 1994, 116, 3121-
3122
1 7 .1. W illner, R. Blonder, and A. Dagan, T . Am. Chem . Soc., 1994, 116, 9365-
9366
18. R.A. Lerner, S.J. Benkovic, and P.G. Schultz, Science, 252, 659-667
19. A.G. Cochran, R. Sugasaw ara, and P.G. Schultz, T . Am. Chem . Soc., 1988,
110, 7888-7890
66
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Files, Darin Joseph
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Core Title
Femtosecond laser studies of biological systems
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Graduate School
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Master of Science
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Chemistry
Degree Conferral Date
1996-05
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University of Southern California
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biology, microbiology,chemistry, physical,OAI-PMH Harvest
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Dalton, Larry R. (
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