2D IR Spectroscopy: Automation with Pulse Shaping and

Application to Amyloid Folding

by

Sang-Hee Shim

A dissertation submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

(Chemistry)

at the

U&IVERSITY OF WISCO&SI&-MADISO&

2008
 i

2D IR Spectroscopy: Automation with Pulse Shaping and

Application to Amyloid Folding

Sang-Hee Shim

Under the supervision of Professor Martin T. Zanni

At the University of Wisconsin-Madison

Two dimensional infrared (2D IR) spectroscopy has proven itself as a unique tool for probing the

structures and dynamics of chemical and biological systems. Now, 2D IR spectroscopy has

progressed to the point of being a ready-to-operate method for general researchers to address

novel scientific questions. For that purpose, this thesis includes three practices: inventing a

technique for mid-infrared pulse shaping; developing an automated method of collecting 2D IR

spectra; applying it to delineate the pathway of amyloid formation. First, we developed a means

to shape femtosecond pulses in the mid-infrared using a germanium acousto-optic modulator (Ge

AOM). The Ge AOM directly modulates the amplitude and phase of mid-IR light between 2~18

µm, producing intense shaped pulses with high resolution and good phase stability. Second, we

combined the pulse shaper with a pump-probe geometry to develop a new method of 2D IR

spectroscopy. The pump-probe geometry simplifies the optical setup and produces properly

phased absorptive lineshapes without additional data processing. The pulse shaper not only

reproduces most conventional methods, but also allows new pulse shapes and phase

combinations to further enhance 2D IR spectroscopy. Moreover, eliminating moving parts

accelerates data collection so that an entire 2D IR spectrum can be obtained in <1 sec. Such

ease of use, versatility and speed of our pulse shaping 2D IR method enables more sophisticated
 ii

and reliable experiments. Finally, we tracked amyloid formation of human Islet amyloid

polypeptide (hIAPP), related to type II diabetes. By continuously scanning 2D IR spectra, we

can follow the hIAPP fibril formation indefinitely by collecting data on-the-fly without re-

initiating the aggregation for each data point. We used isotope-labeling to probe the structural

evolution of six different residues along the 37-residue hIAPP peptide. By separately monitoring

the six residues, we found that the peptides nucleate near the turn of the hairpin, followed by a

propagation of the two parallel β-sheets with the N-terminal β-sheet forming twice as fast as the

C-terminal sheet. The experimental approach provides a detailed view of the assembly pathway

of hIAPP fibril as well as a general methodology for studying other amyloid forming peptides.
 iii

Table of Contents

List of Figures v

Acknowledgements vii

Chapter 1 Introduction 1
1.1 References 11

Chapter 2 Experimental Basics 14

2.1 Introduction 14
2.2 Generation of Amplified Ultrafast Pulses 15
2.2.1 Home-built Ti:Sapphire Oscillator 17
2.2.2 Chirped Pulse Regenerative Amplifier 22
2.3 Generation of Mid-Infrared Pulses 26
2.3.1 Second-Order Nonlinear Optical Interactions 26
2.3.2 Optical Parametric Generation and Difference Frequency Mixing 28
2.3.3 Optical Parametric Amplifier (OPA) 29
2.4 Acknowledgments 33
2.5 References 33

Chapter 3 Femtosecond Pulse Shaping Directly in the Mid-Infrared Using Acousto-

Optic Modulation 34
3.1 Introduction 34
3.1.1 General Theory of Femtosecond Pulse Shaping 36
3.1.2 General Apparatus of Femtosecond Pulse Shapers 37
3.2 Experimental 40
3.2.1 Optical Configuration 40
3.2.2 Triggering and Timing 44
3.2.3 Characterization of Pulse Shapes 47
3.3 Performance of the Mid-IR Pulse Shaper 49
3.3.1 Resolution 49
3.3.2 Efficiency 52
3.3.3 Stability 54
3.4 Applications of the Mid-IR Pulse Shaper 57
3.4.1 Chirp Compensation 57
3.4.2 Pulse Pair Generation 59
3.5 Conclusion 62
3.6 References 63

Chapter 4 Automating 2D IR Spectroscopy Using Mid-Infrared Pulse Shaping 66

4.1 Introduction 66
4.1.1 Conventional Methods of 2D IR Spectroscopy 68
 iv

4.1.2 Principles of Generating 2D IR Spectra from a Pump-Probe Geometry 70

4.2 Experimental 73
4.2.1 Pump-Probe Spectrometer 73
4.2.2 Waveform Generation 74
4.2.3 Signal Detection 77
4.3 Frequency-Domain Method 80
4.4 Time-Domain Method 85
4.5 Phase Incrementing and Phase Cycling 88
4.5.1 Shifting Signal Frequency 89
4.5.2 Removing Background 92
4.5.3 Removing Scatter 95
4.6 Rapid Data Collection 98
4.7 2D Visible Spectroscopy 100
4.8 Conclusion 104
4.9 Acknowledgements 105
4.10 References 106

Chapter 5 Defining the Pathway of Amyloid Formation of human Islet Amyloid

Polypeptide with Residue Specific Resolution 110
5.1 Introduction 110
5.2 Experimental 112
5.3 Kinetics and Morphologies during Amylodosis: Fluorescence/FTIR Spectra and TEM
images 115
5.4 Secondary Structures during Amylodosis: 2D IR Spectra of Unlabeled hIAPP 121
5.5 Residue-Specific Assemblies during Amylodosis: 2D IR Spectra of Isotope-Labeled
hIAPP 125
5.5.1 Six Labeled Residues 125
5.5.2 2D IR Spectra and Kinetics of hIAPP Labeled at Ala 25 127
5.5.3 Reproducibility of the Kinetics 131
5.5.4 Kinetics of Six Labeled Sites 136
5.5.5 Peak positions and Intensities of Isotope-Labels in 2D IR Spectra 145
5.6 Discussion 147
5.7 Conclusion 152
5.8 Acknowledgements 152
5.9 References 153

Appendix I List of Publication 157

 v

List of Figures

Chapter 1
Figure 1.1 1D and 2D IR spectra of hIAPP before and after amylodosis. 8
Figure 1.2 1D and 2D IR spectra of hIAPP labeled at Ala 25 before and after amyloid
formation. 9

Chapter 2
Figure 2.1 Layout of the ultrafast laser system. 16
Figure 2.2 Layout of the home-built Kapteyn-Murnane oscillator. 21
Figure 2.3 Layout of the Spitfire-HPR Ti:Sapphaire amplifier. 25
Figure 2.4 Layout of the home-built optical parametric amplifier. 31
Figure 2.5 Dependence of mid-IR bandwith on OPA optical configuration. 32

Chapter 3
Figure 3.1 Schematic of a femtosecond pulse shaper. 39
Figure 3.2 Experimental setup of a mid-IR pulse shaper using a Ge AOM. 43
Figure 3.3 Triggering and timing scheme of the laser and the shaper. 46
Figure 3.4 Experimental setup for characterizing the shaped pulse. 48
Figure 3.5 Measurement of the shaper resolution. 51
Figure 3.6 Amplitude and position dependence on deflection efficiency. 53
Figure 3.7 Phase stability measurements. 56
Figure 3.8 Compressing the shaped pulse. 58
Figure 3.9 Pulse pairs with various spacing and relative phase. 61

Chapter 4
Figure 4.1 Schematic layouts of 2D IR experiment. 72
Figure 4.2 Experimental setup of the automated 2D IR spectroscopy 79
Figure 4.3 2D IR spectra of W(CO)6 obtained with various shapes of pump pulses. 84
Figure 4.4 Shifting signal frequency of the amide I frequency of NMA in D2O by
incrementing the relative phase. 91
Figure 4.5 Removing transient absorption background from 2D IR specta of NMA in D2O
using phase cycling. 94
Figure 4.6 Removing scatter from 2D IR spectra of hIAPP fiber. 97
Figure 4.7 2D visible spectra of atomic Ru vapour. 104

Chapter 5
Figure 5.1 Morphologies of hIAPP fibers. 118
Figure 5.2 Kinetic studies of hIAPP aggregation using FTIR and ThT fluorescence. 119
Figure 5.3 Concurrent ThT fluorescence and TEM snapshots during hIAPP amyloid
formation. 120
Figure 5.4 2D IR spectra and kinetics curves of unlabeled hIAPP during amylod
formation. 124
Figure 5.5 Structure of hIAPP fibrils obtained from solid state NMR. 126
Figure 5.6 2D IR spectra of hIAPP labeled at Ala25 during aggregation. 129
 vi

Figure 5.7 Kinetics curves of hIAPP labeled at Ala25 during aggregation. 130
Figure 5.8 Reproducibility of four trials on hIAPP labeled at Ala 25. 135
Figure 5.9 Difference 2D IR spectra of mature fibers of the six labeled peptides. 139
Figure 5.10 Difference 2D IR spectra at various aggregation stages of the six labeled
peptides. 140
Figure 5.11 Kinetics data and fits of 6 labeled peptides. 142
Figure 5.12 Comparison of kinetics of 6 labeled peptides. 143
Figure 5.13 Effects of running average. 144
Figure 5.14 Diagonal slices of 2D IR spectra taken for fibers with and without diluting the
label. 146
Figure 5.15 hIAPP aggregation pathway that is consistent with our data. 151
 vii

Acknowledgements

The past five years in graduate school have been the most challenging as well as the most fruitful

time of my life. Not only is Madison distant from my homeland, Korea by half way of the earth

circumference, but also learning and practicing the art of science have been a rare experience that

combines education and amusement. In each step of the period, I have been blessed to have

countless help and support.

First of all, I can not thank enough my advisor, Professor Martin Zanni. He has been

extremely supportive, inspiring, and fun to be with. Whenever I encounter hurdles, personal or

professional, he was always with me, never running out of support and advice. He is enthusiastic

and intuitive in science, but never forgets to enjoy the fun of science. When I decided to study

abroad, my goal was to find a true role model, and I have found him.

In the Zanni group, I have been accompanied by smart, hard-working, and fun individuals.

Eric Fulmer was the very first person whom I worked with. We constructed the home-built

oscillator together and he was always patient and kind to the clueless first-year student. Amber

Krummel and Prabuddha Mukherjee were always supportive and helpful whenever I bother them

with my lack of understanding. Although we never worked on a specific project together, Terry

Ding has been a pleasant and smart character during my days in the laboratory. I worked with

David Strasfeld the most and we constructed the new laser table, pulse shaper and 2D IR

spectrometer together. Dave is not only an uplifting and humorous friend, but also an inspiring

colleague, full of scientific insights. The hIAPP folding project could have never begun without

Yun Ling’s hard work in tackling the finicky sample preparation. Once Wei Xiong joined the

group, he has often been my companion in the laser lab. His intelligence and hard-work will
 viii

definitely benefit him well. Ann Woys and Emily Blanco, smart and sweet ladies of the group,

and I had great girl talks lighting up the darkness of the laboratory. Sudipta Mukherjee and Dr.

Chris Middleton were the last people with whom I worked; although short, I had great time with

them.

I was blessed to have many great collaborators. I acknowledge them in the following

chapters that we collaborated, but I thank them here as well for giving me variety of chances to

explore. I thank Professor David Blank and Dr. David Underwood at the University of

Minnesota who gave us their design of the Ti:Sapphire oscillator and helped us through the

construction. I also thank Professor Niels Damrauer and Eric Grumstrup at the University of

Colorado at Boulders for the collaboration in 2D visible spectroscopy. I never met them in

person, but through numerous emails and phone calls, we could accomplish the experiment in an

efficient way. Finally, I thank Professor Daniel Raleigh and Ruchi Gupta at the State University

of New York at Stony Brook for the collaboration in the labeled hIAPP folding experiment.

They were generous in providing the labeled peptides as well as in educating me with their vast

knowledge in amyloids.

Last, but not least, I sincerely appreciate my family. My parents have always allowed me,

a whimsical character, to find my way by myself, supporting me with all their hearts. Father, I

can no longer see you, but I do know that you are blessing me in heaven. Mother, without your

support, I am sure that I would not have finished graduate school. Brother, you are a great role

model as a scientist as well as a parent. I also thank my new family, my parents in law, who

supported me like their own daughter. Of course, I thank them for giving birth to my husband,

my other half, my companion. I so look forward to sharing the future with you.
 1

Chapter 1
Introduction

The structures and dynamics of proteins have been the focus of study for many researchers to

understand the mechanism of their function. In order for a protein to function, a linear chain of

amino acids must fold into a functional three-dimensional structure, which is uniquely

determined by the sequence of its amino acids through their chemical characters. When a protein

fails to fold into the native form, it misfolds (i.e. incorrectly folds) into an inactive or even toxic

form. Some misfolded proteins form aggregates, known as amyloid, that damage cells and cause

many degenerative diseases (1, 2). Even after folding into their native state, many proteins

undergo conformational changes in response to their environments or upon binding with other

molecules to perform their biological function. For instance, an ion channel controls the flow of

ions by opening and closing its pore in response to a membrane potential or upon binding of a

ligand (3). Many experiments aim to monitor the structural evolution of proteins at sufficient

spatial and temporal resolution to capture the details of their mechanisms. A molecular movie of

amino acids interacting and assembling during protein folding would allow us to answer the

question of how the amino acid sequence translates into the native structure. Following this line

or reasoning, one way to gain insights into the misfolding and toxic activity of amyloids is to

follow the structural changes of a misfolding protein. Understanding the mechanism of ion

gating requires a means to track the structure of the pore at each step of opening and closing of

an ion channel. Therefore, studies associating structure, dynamics and function of proteins need

a method to capture structural details at a sufficient speed so that the individual steps can be

resolved.
 2

One promising method for capturing protein dynamics is two-dimensional infrared (2D

IR) spectroscopy. 2D IR spectroscopy has proven itself as a useful tool to study structures and

dynamics of chemical and biological systems in condensed phases (4-6). The structural

sensitivity comes from vibrational couplings bridging multiple bonds to yield a collective

vibration. The delocalized mode reflects the spatial arrangement of the coupled bonds. In

proteins, backbones arranged in α-helix or β-sheet vibrate in one or two distinct cooperative

motions so that IR absorption from α-helix or β-sheet can be distinguished from one another (7).

To obtain more precise structural information, a local mode (i.e. one residue) can be separated by

labeling the bond with isotopes such that its frequency shifts away from the rest (8, 9). 2D IR

spectroscopy cannot obtain atomic level information like X-ray crystallography or nuclear

magnetic resonance (NMR) spectroscopy. However, 2D IR spectroscopy has ultrafast time

resolution. In contrast, X-ray crystallography captures static structures of crystallized proteins,

while NMR spectroscopy uses radio-frequency pulse sequences to study protein motions taking

place on a time-scale ranging from microseconds to miliseconds (10). 2D IR spectroscopy

obtains dynamical contents from lineshapes that reveal femtosecond dynamics, and from the

evolution of lineshapes that confers picosecond dynamics. The time scale can extend much

farther to seconds or even longer by using 2D IR spectroscopy as a camera to take snapshots of

an evolving system. In fact, snapshots can be taken by other techniques, but each 2D IR

snapshot reflects its unique ultrafast dynamics at the very moment when the snapshot is captured.

Moreover, 2D IR spectra can be collected from virtually any kind of sample, as demonstrated by

linear IR spectroscopy, including membranes and fibers that other structure-resolving tools have

difficulty probing. These capabilities of 2D IR spectroscopy for protein dynamics have been
 3

demonstrated in various problems, including sub-milisecond protein folding (11, 12),

membrane peptide structure assessment (13), proton channel gating (14) and amyloid formation

(15).

Since Hamm, Lim and Hochstrasser collected the first 2D IR spectrum in 1998 (16), the

field of 2D IR spectroscopy has focused in advancing the technique and demonstrating its

capabilities. Now the field faces a new phase calling for a turnkey equipment that allows many

physicists, chemists and biologists to apply 2D IR spectroscopy to novel scientific questions. So

far, its complicated implementation has restricted the number of researchers to a few dedicated

groups. A ready-to-use, reliable spectrometer will enable 2D IR spectroscopy to provide new

insights on variety of topics including protein dynamics. To initiate the new phase of 2D IR

spectroscopy, we invented an automated method of collecting 2D IR spectra using a mid-IR

pulse shaper and applied the method to delineate the pathway of amyloid formation.

Among many possible applications, amyloid formation is of particular interest, not only

because it is well suited for demonstrating the unique capabilities of our method, but because it

will also propose potential drug targets for many human diseases including diabetes,

Altzheimer’s and Parkinson’s disease. More than 20 diseases are known to associate with

proteins misfolding into amyloid fibers (1, 2). Abnormal accumulation of amyloid fibers is

commonly observed in diseased tissues. Recently, evidence pointing to the partially folded

intermediates as cytotoxic entities has accumulated, which is steering the focus of amyloid

research from mature fibers to intermediates (17-19). Identifying the structure and defining the

evolution of such cytotoxic species is vital to design inhibitors that intervene the peptide’s

damaging action. Studies containing claims of critical intermediates have provided little

structural information, mostly because it is extremely difficult to obtain both structural and
 4

kinetic information simultaneously during aggregation. X-ray crystallography and solid-state

NMR spectroscopy do not have sufficient time resolution to track the structural changes, not to

mention that they can seldom work with aggregating systems. Circular dichroism (CD), Fourier-

transform infrared (FT IR) and fluorescence spectroscopy have sufficient time-resolution, but

only provide a low resolution in structure (20, 21). Moreover, studies of amyloid with CD or FT

IR spectroscopy often encounter difficulties on finding a unique fit when deconvoluting the

congested spectrum into features for β-sheet, random coil and α-helix. Resolving the structural

evolution of the cytotoxic intermediates requires a technique with sufficient time and structural

resolution at the same time.

2D IR spectroscopy is well-suited to study amyloid formation due to its better structural

resolution than other 1D spectroscopies including CD and FT IR spectroscopy. The delocalized

modes of secondary structures in 1D IR spectra are spread over two dimensions so that each

secondary structure can be assigned to a unique spectral component. For instance, shown in Fig.

1.1 are 1D and 2D IR spectra of hIAPP (human Islet amyloid polypeptide), associated to type II

diabetes, before and after amyloid formation (15, 22). The amide I band of a peptide at

1600~1700 cm-1 can be approximated as a sum of carbonyl stretches in the backbone and is

sensitive to the secondary structure. When freshly dissolved in buffer, the peptides begin as

monomers in random coil (Fig. 1.1(a)). When carbonyls in a peptide are randomly distributed,

thus randomly coupled, the frequencies of the carbonyl stretches are broadly distributed.

Therefore, the FT IR spectrum in Fig. 1.1(c) gives a broad peak centered at 1644 cm-1; the 2D IR

signature in Fig 1.1(e) is an out-of-phase doublet elongated along the diagonal. In 2D IR spectra,

each vibrational mode produces a pair of peaks that probe two different sequences of interactions

between the mode and the light pulses; one involves only with the fundamental band; another
 5

includes the overtone, thus shifts the peak along ωprobe. After the amyloid fibril mature, the

fibril from hIAPP comprises stacks of hairpins with a turn connecting two β-sheets, one of which

tails to a random coil (Fig. 1.1(b)) (23). In a β-sheet where the carbonyls form a pleated sheet in

two dimensions, two delocalized modes are allowed, antisymmetric and symmetric stretches.

The most obvious feature in the FT IR spectrum of fibers is the antisymmetric mode at 1618 cm-1

(Fig. 1.1(d)) that gives the dominant doublet in the 2D IR spectrum (Fig. 1.1(f)). Note that the

major feature at 1618 cm-1 is more prominent in the 2D spectrum than in the 1D spectrum

because 2D IR intensities are more dependent on the transition dipole strengths than 1D IR

intensities are. Moreover, the 2D IR spectrum gives a unique feature of the vertical crosspeak

along ωpump from 1618 to 1673 cm-1 that connects the two overtone peaks of β-sheet

antisymmetric and symmetric mode. The crosspeak indicates strong coupling between the two

modes, which is expected from the fact that the two modes stem from the same carbonyls

arranged in β-sheet. As seen above, it is common that spectra in one dimension suffer from

congested features because all the secondary structures absorb similar wavelengths. 2D IR

spectra give better distinction between the spectral features from different secondary structures

by highlighting features from ordered structures and by providing unique features of cross peaks

(15).

The structural resolution of 2D IR spectroscopy can be further improved to a single

residue by using site-specific isotope-labeling (13). When a backbone carbonyl of a residue is

13 18
labeled with C O, its amide I frequency is shifted by ~70 cm-1 so that the single residue is

separated from the rest of the protein with 12C16O (9). Shown in Fig. 1.2 are the 2D IR spectra

taken from hIAPP labeled at Ala 25 before and after aggregation (24). The spectral features

from the unlabeled portion of the peptide (dotted box in Fig. 1.2(c-d)) are similar to the ones
 6

described before (see Fig. 1.1(e-f)). New features located at ωpump=ωprobe=1570~1580 cm-1

belong to the 13C18O-label at Ala25 (solid box in Fig. 1.2(c-d)). The spectral feature of the label

before aggregation is very broad and weak (Fig. 1.2(c)) because the disordered monomers

distribute the label randomly in space (black circles in Fig. 1.2(a)). In the mature fibril

composed of parallel stacks of peptides, the isotope-labels at Ala25 form a column along the

fibril long axis (black circles in Fig. 1.2(b)). The linear alignment strongly couples the labeled

carbonyls so that the label feature narrows and intensifies in the 2D IR spectrum (solid box in

Fig. 1.2(d)). In fact, the fibril spectrum in Fig. 1.2(d) is a difference spectrum from the first and

the last spectrum of a single aggregation experiment. The subtraction highlighted the spectral

changes so that the weak feature from a single residue became noticeable. Even a crosspeak was

detected between the label at Ala 25 and the β-sheet antisymmetric stretch from the rest of the

peptide (follow the arrow in Fig. 1.2(d)), indicating the proximity of the label to the β-sheets.

Therefore, a single residue can be tracked during amyloid formation with isotope-edited 2D IR

spectroscopy.

Conventional methods of 2D IR spectroscopy do not offer sufficient time resolution for

amyloid studies. Those methods spend tens to hundreds of minutes to collect a single spectrum,

which is too long considering that amyloid formation is often complete within hours. Transient

2D IR spectroscopy can be collected with time resolution of picoseconds (12), but it requires re-

initiating the reaction hundreds of times to average a signal at each point of a 2D IR spectrum

(25). However, the transient method will not work for amyloid aggregation, which is infamous

for its poor reproducibility. This is because the kinetics of amyloid formation is extremely

sensitive to the composition and purity of the sample (26, 27), and to the conditions of initiation

and incubation (28). To track a single aggregating sample with 2D IR spectroscopy, we need a
 7

rapid means of scanning time delays between multiple pulses that generates and samples the 2D

IR signal. In the conventional methods of 2D IR spectroscopy, multiple pulses are generated by

splitting one IR light into multiple beam paths. Their time delays are scanned by controlling the

path lengths with translational stages that spend a deadtime of 50-100 ms for each translation

(29). Therefore, a new means of generating pulse sequences without spending time on moving

parts will speed up the data collection so that 2D IR spectra can be collected on-the-fly while the

peptides aggregate into amyloid fibers.

As in NMR spectroscopy where radio-frequency pulse shaping generates pulse sequences,

optical pulse sequences can be generated programmably by femtosecond pulse shaping.

Recently, we developed a mid-infrared pulse shaper that can directly shape mid-IR light (30, 31).

Our shaper spends only 10 microseconds to load a new pulse shape, which is even faster than the

repetition rate of most lasers used in 2D IR spectroscopy. With this powerful tool in hand, we

developed an automated version of 2D IR spectroscopy combining the mid-IR pulse shaper with

a pump-probe geometry (22). While the pump-probe geometry simplifies the setup into two

beams, the pulse shaper generates pulse pairs with accurate phase and time delay in a speed rapid

enough to use every laser shot. When using 1 kHz laser systems, an entire 2D IR spectrum can

be collected <1 sec. Thus, the speed of the method would encourage many researchers to use 2D

IR spectroscopy in their kinetics studies. In fact, the rapid data collection allowed us to track the

amyloid formation from a single hIAPP sample with a sufficient time resolution to follow the

kinetics (15).

Chapter 3 discusses the technology we invented, mid-infrared pulse shaping. The mid-IR

pulse shaper uses germanium acousto-optic modulator (Ge AOM), a programmable diffractive

optic whose efficiencies and phases across the crystal aperture are modulated by the amplitude
 8

and phase of the acoustic wave passing through the crystal (30, 31). The Ge AOM that shapes

mid-IR at 2~18 µm has efficiency and resolution on par with visible modulators. The optical

alignment and electronics configurations are discussed in detail for future reconstruction. The

performance and simple applications are also presented.

Using the mid-IR pulse shaper, we developed a method of collecting 2D IR spectra as

Chapter 4 will describe. The method combines the mid-IR pulse shaper with a pump-probe

geometry (22). The two-beam geometry simplifies the optical setup; the programmable

generation of pulse sequence makes data collection straightforward and instantaneous. Chapter

4 details the optical and electronic configuration for the rapid-scanning method that continuously

cycles a set of waveforms assigning time delays and synchronously detects the 2D IR signal.

Various pulse shapes and phase combinations are demonstrated along with rapid data collection

to further enhance 2D IR spectroscopy in ways that only pulse shaping can accomplish.

Finally, Chapter 5 presents the application of the automated 2D IR spectroscopy to

defining the pathway of amyloid formation of hIAPP (15, 24). Site-specific isotope-labeling was

used to selectively probe six residues evenly spaced along the 37-residue peptide. Since the

fibril consists of parallel β-sheets crossing the fiber axis, isotope-labeled carbonyls form a linear

column along the fiber long axis. Thus, the kinetics curve of each residue reflects the timing of

the column formation during aggregation. By acquiring kinetics curves of the six residues

separately, the order of amyloid assembly was delineated; the aggregation nucleates around the

turn, and then propagates along the final hairpin. The experimental approach combining the

automated 2D IR spectroscopy and isotope-labeling provides not only a detailed view of the

aggregation pathway of hIAPP, but also a general methodology for studying self-assembling

proteins to develop inhibitors for disease-causing amyloids and to better design biomaterials.
 9

Figure 1.1 Amyloid formation of hIAPP. The monomers in random coil (a) aggregate into

fibrils rich in β-sheets (b). 1D IR and 2D IR spectra of hIAPP are presented before (c and e) and

after aggregation (d and f). The left column shows structure and spectra of the monomers before

aggregation and the right column contains those for mature fibrils. The top row shows a

schematic of structures exemplified with four peptides. The center row and the bottom row

display the 1D and 2D IR spectra, respectively.

 10

Figure 1.2 Amyloid formation of hIAPP labeled at Ala 25. The monomers in random coil

(a) aggregate into fibrils rich in β-sheets (b). 2D IR spectra of hIAPP labeled at Ala 25 are

presented before (c) and after aggregation (d). The left column shows structure and spectra of

the monomers before aggregation and the right column contains those for mature fibrils. The top

row shows a schematic of structures exemplified with four peptides labeled with black circles

representing the isotope-label at Ala 25. The bottom row presents the 2D IR spectra with solid

black boxes highlighting the label feature, dotted boxes enclosing the unlabeled features, and a

black arrow pointing the crosspeak between the label and the β-sheet feature from the remainder

of the peptide.
 11

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measured by femtosecond nonlinear-infrared spectroscopy. J. Phys. Chem. B
102(31):6123-6138.

17. Kayed R, Head E, Thompson JL, McIntire TM, Milton, S. C. C, C. W,, & G. GC (2003)
Common structure of soluble amyloid oligomers implies common mechanism of
pathogenesis. Science 300:486-489.

18. Kagan B (2005) Oligomers and cellular toxicity in amyloid proteins: The beta sheet
conformation and disease. (WILEY-VCH Verlag GmbH & Co., Weinheim).

19. Silveira JR, Raymond GJ, Hughson AG, Race RE, Sim VL, Hayes SF, & Caughey B
(2005) The most infectious prion protein particles. ature 437:257-261.

20. Jayasinghe SA & Langen R (2005) Lipid membranes modulate the structure of islet
amyloid polypeptide. Biochemistry 44(36):12113-12119.

21. Knight JD, Hebda JA, & Miranker AD (2006) Conserved and cooperative assembly of
membrane-bound alpha-helical states of islet amyloid polypeptide. Biochemistry
45(31):9496-9508.

22. Shim S-H, Strasfeld DB, Ling YL, & Zanni MT (2007) Automated 2D IR spectroscopy
using a mid-IR pulse shaper and application of this technology to the human islet amyloid
polypeptide. Proc. atl. Acad. Sci. U.S.A. 104(36):14197-14202.

23. Luca S, Yau WM, Leapman R, & Tycko R (2007) Peptide conformation and
supramolecular organization in amylin fibrils: Constraints from solid-state NMR.
Biochemistry 46:13505-13522.

24. Shim S-H, Gupta R, Ling YL, Strasfeld DB, Raleigh DP, & Zanni MT 2D IR
spectroscopy and isotope labeling defines the pathway of amyloid formation with residue
specific resolution. Submitted.

25. Bredenbeck J, Helbing J, Kolano C, & Hamm P (2007) Ultrafast 2D-IR Spectroscopy of
transient species. Chemphyschem 8(12):1747-1756.

26. Padrick SB & Miranker AD (2002) Islet amyloid: Phase partitioning and secondary
nucleation are central to the mechanism of fibrillogenesis. Biochemistry 41(14):4694-
4703.

27. Ruschak AM & Miranker AD (2007) Fiber-dependent amyloid formation as catalysis of

an existing reaction pathway. Proc. atl. Acad. Sci. U.S.A. 104(30):12341-12346.
 13

28. Petkova AT, Leapman RD, Guo ZH, Yau WM, Mattson MP, & Tycko R (2005) Self-
propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science
307(5707):262-265.

29. Nee MJ, McCanne R, Kubarych KJ, & Joffre M (2007) Two-dimensional infrared
spectroscopy detected by chirped pulse upconversion. Opt. Lett. 32(6):713-715.

30. Shim S-H, Strasfeld DB, Fulmer EC, & Zanni MT (2006) Femtosecond pulse shaping
directly in the mid-IR using acousto-optic modulation. Opt. Lett. 31(6):838-840.

31. Shim S-H, Strasfeld DB, & Zanni MT (2006) Generation and characterization of phase
and amplitude shaped femtosecond mid-IR pulses. Opt. Express 14(26):13120-13130.
 14

Chapter 2
Experimental Basics

2.1 Introduction to Femtosecond Laser Spectroscopy

Since the early 1990s, ultrafast lasers have created revolutions in physics and chemistry by

opening new fields, including nonlinear optics and time-resolved spectroscopy (1). The term

“ultrafast” comes from their output pulse duration of femtoseconds or picoseconds (2).

Scientists have applied ultrafast lasers to monitor dynamics occurring on time scales of

femtoseconds or picoseconds. Examples of such phenomena are femtosecond dynamics in

condensed phase including electrons in solids (especially, semiconductors) and chemical

reactions. For chemists, the dynamics of molecules in liquids is of particular interest because

most chemistry performed in the laboratory occurs in the liquid phase. In liquids, vibrations of

chemical groups and molecules occur in femtoseconds and relax in picoseconds. Thus, a laser

system that can investigate time scales sufficiently short to observe femto/picosecond dynamics

has been the tool of choice for many researchers in physical chemistry.

The discovery of mode-locking in lasers with titanium doped sapphire (Ti:Sapphire)

crystals gave birth to modern ultrafast lasers. Further technical advances in amplification (3) and

nonlinear crystals (4, 5) have expanded the spectrum of ultrafast lasers from soft X-rays to

Terahertz. This chapter aims for describing our way of generating femtosecond pulses in the

mid-infrared to perform 2D IR experiments in the following chapters. Since there exists no

femtosecond lasers directly emit mid-infrared light, 800-nm light from a mod-locking laser is
 15

amplified by a regenerative amplifier, then transferred to the mid-infrared via optical

parametric amplification (OPA) combined with difference frequency mixing.

2.2 Generation of Amplified Ultrafast Pulses

The femtosecond pulses at 800 nm should have sufficiently high energy in 100 µJ to mJs to

pump an OPA generating mid-IR with microjoules of energy. Most mode-locking lasers cannot

produce such a high power, so a regenerative amplifier is used such that femtosecond pulses

from a mode-locking laser gain more power from a pulsed green laser with high power (tens of

watts). In our laboratory, a commercially available ultrafast laser system from Spectra-Physics

generates sub-45fs pulses at 800 nm with 2-mJ of energy. Shown in Fig. 2.1 is the ultrafast laser

system including a home-built Ti:Sapphire oscillator (88 MHz) pumped with a Millenia

Nd:YVO4 continuous wave (CW) laser as well as a Spitfire-HPR Ti:Sapphire regenerative

amplifier pumped with a Evolution-30 Nd:YLF pulsed laser (1 kHz). The oscillator generates

femtosecond pulse trains by Kerr-lens mode-locking, and then seeds the regenerative amplifier to

gain high power via chirped pulse amplification. Since the oscillator pulses spontaneously, all

electronics devises are synchronized to a reference pulse at 88 MHz from a photodiode

monitoring the oscillator. For instance, the Evolution-30 is triggered by a 1 kHz TTL pulse from

a divider circuit that divides the 88 MHz reference by 88,000.

 16

Figure 2.1 Layout of our ultrafast laser system. The Millenia Nd:YVO4 continuous wave

laser pump the home-built Ti:Sapphire oscillator to generate 800 nm <25fs laser pulse. The

pulse train from the oscillator is seeded to the Spitfire-HPR Ti:Sapphire regenerative amplifier,

which is pumped by the Evolution-30 Nd:YLF ns-pulsed laser. Note that the Evolution is

synchronized to the oscillator by an external trigger generated from a divider circuit that

produces a 1 kHz TTL pulse synchronized to the 88 MHz pulse train from a photodiode

monitoring the oscillator. The output of the laser system is <45 fs pulses with 2 mJ energy per

pulse at 800 nm.

Millenia home-built
Nd:YVO4 Ti:Sapphire
CW laser CW Oscillator
532nm
2.4W
88MHz
divider
circuit
800nm, <25fs,
1kHz ~2 nJ/pulse
TTL

Evolution-30 Spitfire-HPR 800nm, <45fs

Nd:YLF Ti:Sapphire
2 mJ/pulse
ns laser 1kHz Regenerative Amplifier 1kHz
532nm
23 mW
 17

2.2.1 Home-built Ti:Sapphire Oscillator

In most ultrafast laser systems, ultrashort pulses are spontaneously generated from a Ti:Sapphire

laser that is pumped with green continuous wave light. The Ti:Sapphire crystal exhibits the

optical Kerr effect where an intense optical field causes a variation in the refractive index of the

crystal in proportional to the local intensity of the light (6);

n = n0 + n2 I (2.1)

where n is the refractive index of the material, n0 and n2 are the zeroth and second-order term,

and I is the intensity of the incident electric field. As a pulse propagates through the crystal, the

intensity dependence of the refractive index induces the “self-focusing” of the femtosecond

pulsed mode; i.e. the peak portion of the pulse is focused more than the tail portion with lower

intensity. As a result, the peak intensity of self-focusing pulse keeps increasing as the pulse pass

through the crystal. This divergence of the beam profiles leads the pump laser to associate

preferentially to the femtosecond mode, which in turn gains higher power than the continuous

wave mode. Thus, once the laser mode-locks, the mode-lock mode remains and dominates,

when the laser is aligned properly. In practice, the laser does not start mode-locking by itself and

lases in the continuous wave mode when turned on. A “seed” noise spike, commonly produced

by vibrating one of the optics in the cavity, is given to buildup as mode-locking.

We built an oscillator (Fig. 2.2) based on the design of Kapteyn and Murnane which is

capable of producing pulses as short as 11 fs (2). Our design uses a short Ti:Sapphire crystal

(Saint-Gobain, 0.25% doping, 3 mm diameter) that has a path length of 4 mm. The crystal is

pumped by a Nd:YVO4 laser (Spectra-Physics Millenia), focused by a lens to focus the beam

into the crystal. The crystal sits in a copper, water cooled mount between a pair of dichroic
 18

concave mirrors. The crystal, curved mirrors, and lens are mounted on translational stages

which are mounted to a baseplate predrilled at correct positions. A pair of prisms in conjunction

of a retroreflector compensates dispersion from the crystal and the cavity optics. Following is a

basic alignment procedure for constructing the oscillator.

(1) Align the green pump beam. First, construct a periscope to rotate the polarization of the

pump beam in vertical polarization. Second, align the turning mirror for the pump beam

through two irises mounted to the baseplate. Third, place the pump lens and adjust it to

keep the pump beam on the two irises. Fourth, place the curved mirror close to the lens

and adjust it so that the pump beam passes through the center of the curved mirror.

(2) Place the Ti:Sapphire crystal. First, mount the crystal into the custom-machined, water-

cooled, copper crystal mount after greasing the crystal with thermal grease. Second,

rotate the mount so that the crystal reflects the green pump at minimum intensity (i.e. at

Brewster angle).

(3) Assemble the curved mirror away from the pump lens similarly to another curved mirror.

(4) Build a small laser cavity by placing the end mirror and output coupler as close as

possible to the curved mirrors. While pumping the crystal, align the end mirror and

output coupler for lasing by overlapping the fluorescence spots. Optimize power and

mode (round) by alternately tweaking horizontal and vertical knobs on both end mirrors,

and translating the lens, curved mirrors, and crystal.

(5) Extend the cavity gradually. Do this by moving the cavity mirrors, one at a time, while

maintaining lasing. Stop every few inches and optimize the laser power by moving the

curved mirrors and crystal, and by adjusting the end mirror. Usually the laser mode-

locks best when the extension is done asymmetrically, with the arm containing prisms
 19

much longer than the other arm. The distances between optics are denoted in the Fig.

2.2 to obtain a repetition rate of 88 MHz.

(6) Assemble the first prism. Once the arm containing the end mirror extends out of the

potential position of the first prism, replace the end mirror with a gold-coated plane

mirror, while keeping lasing. Insert the first prism by grazing a bit of the laser beam off

the apex of the prism. Rotate the prism for minimum deviation of this light. Retro-

reflect the diffracted beam from the prism using the end mirror. Once the beam is

retracting itself, translate the prism until the laser is barely lasing. Optimize the lasing

power from a cavity containing the first prism and the end mirror. Extend the cavity

further as in Step 5

(7) Assemble the second prism in the same way in Step 6. Then, extend the cavity as in

Step 5 until the cavity length becomes as designed. Once the two prisms are placed,

place a card in front of the gold mirrors paired with each prism to prevent additional

lasing. These gold mirrors are used only when the laser stop lasing and need to shorten

the cavity for alignment.

(8) Pick up the continuous wave laser power. Adjust the end mirror, the output coupler, the

lens and the curved mirrors to get round continuous wave mode and maximum power.

With 2.4W pump power, we usually get >300 mW.

(9) Now the laser is ready to mode-lock. To detect the mode-locking, we use a spectrometer

and a fast photodiode. When the cavity lases in continuous wave, a very narrow

spectrum is observed on the spectrometer and a constant offset is observed by the

photodiode. When the laser mode-locks, the spectrum broadens and the photo diode

detects pulse trains. First, align the laser for maximum continuous wave power and for a
 20

round TEM00 mode. Translate the prisms so that the beam is within 1-2mm from

edge. Then move the curved mirror away from the pump lens towards the crystal until

the output beam looks oval, vertically. When the cavity is ready for mode-locking, the

spectrum will get “jumpy” and the photodiode signal will become noisy. Pushing the

translational stage mounting the curved mirror away from the pump lens should cause

the laser to self-mode-lock.

(10) Once the laser mode-locks, translate the curved mirror until the laser mode-locks easily.

Adjust the translation of the two prisms (varying the amount of glass) for a desired

center frequency and bandwidth. Then, tweak up the mode-locked power with the end

mirrors. When aligned properly, the mode-locked laser has perfectly round mode and

more power than in continuous wave mode (up to 50% more), while the continuous

wave mode is oval and blurred. 150~200 mW of mode-locked power is sufficient to

seed the regenerative amplifier.

(11) Often, there is some continuous wave light co-lasing with the mode-locked light, which

produces a sharp and tall spike in the spectrum. The best way to get rid of this is to turn

down the pump power. You can suppress the continuous wave by using an aperture

clipping the beam inside cavity.

 21

Figure 2.2 Layout of a Kapteyn-Murnane oscillator including a Ti:Sapphire lasing

medium, a continuous wave pump source, curved mirrors to pass the pump light (532 nm) and to

reflect the emitted light (800 nm), a high-reflective end mirror, an output coupler with 12%

transmission, and a pair of prisms to compensate dispersions inside the cavity.

TM
Periscope Nd:YVO4
Curved TM Pump Laser
beam Mirror
block Curved
Mirror Pump 2.4W
Ti:Sapphire Lens
CW
800 nm
15.3o
<25 fs
58.8 cm 15.3o TM
88MHz Prism
Output 30 cm Au
Coupler
58.3 cm
Prism
Au 12.7 cm
End
Mirror

Optics

Ti:Sapphire Brewster cut, 3 mm diameter, 4 mm length, 0.25% doping (Saint-Gobain)

Curved Mirror plano-concave, radius of curvature = 100 mm (Layertec #101241)

anti-reflective (AR) (0º, <5%) coated for 532 nm,
high-reflective (HR) (0º, >99.8%) coated for 720-920 nm

Output Coupler plane, partial-reflective (88±2.5%) coated for 720-890 nm

(Newport #10B20-010C.20)

End Mirror plane, HR (0º, >99.9%) coated for 710-880 nm (Layertec #101241)

Prism Isosceles Brewster prism, fused silica (CVI # IB-124-69.1-UV)

Au Gold-coated plane mirrors for aligning the cavity

Pump Lens plano-convex lens, radius of curvature = 51.5 mm

AR coated for 532 nm (CVI #PLCX-15.0-51.5-C-532)

TM Turning mirror, HR (45º, >99%) coated for 532 nm (Layertec #100765)

 22

2.2.2 Chirped Pulse Regenerative Amplifier

Ti:Sapphire oscillators produce a few nJ per pulse, which is not enough to pump an OPA to

generate mid-IR light with a few µJ of energy to perform 2D IR experiments. We therefore

amplify the pulses using Spitfire-HPR from Spectra-Physics that includes a stretcher, a

regenerative amplifier, and a compressor in conjunction with a high power pump laser at 532 nm

running at 1 kHz as well as fast electronics for accurate timing the amplification (3). The

Spitfire-HPR uses Ti:Sapphire crystal as an amplifying medium due to its high saturation

fluences and a large gain-bandwidth. Since intense beams can cause the crystal to self-focus the

beam destructively or to get damaged, it is necessary to limit the intensity present in amplifiers.

The Spitfire-HPR overcomes this obstacle by using chirped pulse amplification. Briefly, a

femtosecond pulse from an oscillator is stretched to reduce its peak power significantly. This

stretched pulse with low peak power is then amplified, so that damage of the laser rod is much

less likely to happen. Following amplification, the pulse is recompressed to near its original

duration. The Spitfire-HPR (shown in Fig. 2.3) stretches the femtosecond seed pulses by as

much as 10,000 times and amplifies the pulse energy up to 106 times.

Pulse stretching and compression use diffraction grating (or prism as in the oscillator) to

disperse the incident beam so that different frequencies travel through different path lengths.

The grating and retroreflectors can be configured such that the bluer frequency components have

to travel longer distance through stretcher than the redder ones. Thus, the redder the frequency,

the earlier it exits the stretcher, and vice versa. Therefore, the pulse is stretched in time with

redder frequencies traveling in the front and bluer frequencies retarded in the back. The

stretcher in Spitfire-HPR (Fig. 2.3) uses a single grating and a multi-passed beam configuration

that the incident beam passes the grating four times to reconstruct the stretched beam spatially.
 23

Pulse compressor, in essence, reverses what pulse stretcher does. The gratings and mirrors are

arranged so that the bluer frequencies travel the shorter path to catch up with the redder

frequencies. Spitfire-HPR uses an automatic translational stage on a pair of retroreflectors (see

Fig. 2.3) to fine-tune the compression.

The stretched seed is sent to the regenerative amplifier cavity to gain high energy up to

106 times in Spitfire-HPR. In our system, 2 nJ of seed is amplified to 2 mJ. In principle, the

regenerative amplifier controls polarization to confine a single pulse out of seed pulse train, to

amplify the pulse to a high energy level, and then to dump the output from the cavity. The

amplification take place in the laser rod pumped by a high power ns-pulse from an Nd:YLF laser

(23 W, for us). A single pass through the rod only gives a factor of 3-4 amplification; however,

the regenerative amplifier allows the pulse pass through the crystal multiple times to gain much

higher amplification. The Spitfire-HPR use a pair of Pockels cells, which are programmable λ/4

waveplates, to control the multi-pass. When both of the Pockels cells (PC#1 and #2 in Fig. 2.3)

are off, the seed oscillates only once and gets little amplification. The amplification process

begins as the PC#1 is turned on, following the activation of the Q-switch on the pump laser. A

λ/4 waveplate placed near PC#1 orthogonally rotates the polarization of the double-passing beam

(i.e. λ/2 rotation). Thus, when the PC#1 is off, the beam transmits the resonator optics.

However, when the PC#1 is activated to serve as additional λ/4 waveplate, it negates the effect

of the neighboring λ/4 waveplate, trapping the pulse in the resonator. After the pulse makes a

number of round trips (usually ~20), the PC#2 is activated so that the pulse polarization is

orthogonally rotated after double-passing the Pockels cell. Then, a thin-film polarizer placed

after PC#2 expels the multi-passed pulse from the resonator. During the trapping period in the

resonator, the seed pulse multi-passes the rod with a gain of 106.
 24

Correct timing of switching the Pockel cells is essential for the regenerative amplifier

to perform. Multiple output pulses can be generated by an error of a couple of nanoseconds. To

guarantee admission of a single pulse to the resonator, the Pockels cell must be switched

synchronously to the mode-locked pulse train. For this aim, a photodiode monitors the pulse

train out of the mode-locking oscillator to generate a reference signal. In addition, the switching

phase should be fine-tuned to prevent additional output pulse. The Spitfire-HPR provides the

“Synchronization and Delay Generator” (SDG) module containing the synchronizing and phase

adjusting electronics. The SDG module is triggered by a TTL pulse that is provided from the

pump laser, Evolution-30, which is synchronized to the oscillator by a 1 kHz reference from a

divider circuit that divides the 88 MHz reference by 88,000 times. The SDG produces trigger

pulses to control two Pockel cells with adjustable delays. To eject the amplified output after

sufficient round trips, the second Pockel cell is activated approximately 200 ns after the first

Pockel cell switches.

 25

Figure 2.3 Layout of the Spitfire-HPR Ti:Sapphaire amplifier with pulse stretcher and

compressor.

Seed

Compressor

Output

Stretcher
PC#2

λ/4 PC#1
Pump
Regenerative Amplifier

Optics

plane mirror curved mirror grating

lens, convex lens, concave waveplate or polarizer

PC#_
Pockels cell
 26

2.3 Generation of Mid-Infrared Pulses

An optical parametric amplifier (OPA) transfers the Spitfire output at 800 nm to the mid-infrared.

The frequency conversion is accomplished in two steps: optical parametric generation using β-

barium borate (BBO), followed by difference frequency mixing with AgGaS2 crystal, both of

which are second-order nonlinear processes (7, 8).

2.3.1 Second-order Nonlinear Optical Interactions

The macroscopic polarization of a medium, P(t), in response to a light illumination can be

written as a Taylor series of the electric field E:

P (t ) = χ (1) E (t ) + χ ( 2) E (t ) 2 + χ (3) E (t ) 3 + L . (2.2)

The χ(1) is the first-order susceptibility describing linear optics; The χ(2), χ(3) … are the second

and third-order susceptibilities that account for nonlinear optical effects (4). With a sufficiently

weak illumination, the material linearly responds to the electric field. The nonlinearity is

observed when the electric field is sufficiently intense to break the linear assumption, mostly

when ultrashort pulses are illuminated. Note that the optical Kerr effect described in Eq. 2.1 is a

third-order nonlinear process and 2D IR spectroscopy will be discussed in terms of third-order

nonlinear response. Here, the second-order susceptibility, χ(2), accounts for optical parametric

generation as well as difference frequency mixing.

Materials with large χ(2) are used to generate new frequencies via parametric effects

involving three photons. The three photons obey energy conservation by linearly combining the

three frequencies as well as momentum conservation by linearly combining the three wave
 27

vectors. Let us assume that the three photons have frequencies of ω1, ω2 and ω3 with an order

of ω1< ω2< ω3 and have wavevectors k1, k2, k3, respectively. Then,

ω3 = ω1 + ω 2 (2.3),

k 3 = k1 + k 2 (2.4),

which are called as phase-matching conditions.

Nonlinear crystals are cut and arranged to satisfy phase-matching conditions for a desired

scheme of frequency conversion (5). Most of such nonlinear crystals are birefringent material,

whose indices of refraction depend on the polarization and direction of the light passing through.

Among three axes of the birefringent crystal, one axis has different refractive index, thus is

called as “extraordinary” axis; the other two axes are called “ordinary” axis. The polarization of

the lights and the crystal orientation are designed to obey the phase-matching condition. Among

several schemes of choosing polarizations, type I and type II are most common. In type I phase-

matching, the lights at frequencies of ω1 and ω2 (i.e. two lower frequencies) have the same

polarizations. In type II phase-matching, the lights at frequencies of ω1 and ω2 are orthogonally

polarized. Once a crystal is chosen, the angle between the incident lights and the axes of the

crystal determines the frequencies of outputs from nonlinear crystals by fulfilling phase matching

conditions. Thus, the frequency conversion via nonlinear crystals are tunable by rotating the

crystal.
 28

2.3.2 Optical Parametric Generation and Difference Frequency Mixing

Of many parametric effects, our OPA utilizes optical parametric generation and difference

frequency mixing. The BBO crystal generates a signal and an idler light in near-infrared from

the pump pulse at 800 nm via optical parametric generation obeying the phase matching

conditions of

ω pump = ωsignal + ωidler , (2.5)

k pump = k signal + k idler . (2.6)

From the signal and idler lights are generated, the following AgGaS2 crystal produces mid-

infrared light at 2 to 18 µm, fulfilling the following conditions:

ω MIR = ωsignal − ωidler ; (2.7)

k MIR = k signal − k idler . (2.8)

Note that the wave vector depends on the refractive index of the crystal, i.e.

n(ω )ω
k= . (2.9)
c

Then, a nonlinear crystal can be designed from the mathematical representation of the refractive

indices for extraordinary and ordinary axes of the crystal regarding the frequencies and the

polarizations of the incident and outgoing beams, as well as a desired angle of the crystal.

We chose the cutting angles and types of BBO and AgGaS2 from the frequencies and

polarizations of the given pump at 800 nm and the desired mid-IR at 5 or 6 µm. Also, the crystal

orientation was set to be normal to the table so that the path length of the incident beam through

the crystal, thus material dispersion, kept minimal. Once the OPA built, the mid-IR frequency is
 29

tuned by rotating AgGaS2 crystal. Also, the signal and idler frequencies mixed at AgGaS2 are

tuned by rotating the BBO crystal.

2.3.3 Optical Parametric Amplifier (OPA)

The layout of our optical parametric amplifier (OPA) is shown in Fig. 2.4. The design of the

OPA is similar to others (8, 9). The 2 mJ 45 fs transform limited pulses from the Spitfire is

equally divided by two with a 50% beamsplitter to pump two OPAs. Two OPAs have similar

layout and each are pumped with 1 mJ of 800 nm pulses. Downconversion of the 800 nm into

signal and idler pulses (140 µJ) takes place in a type II BBO (θ=25.9°) crystal in two stages with

collinear alignment. Mid-IR pulses are generated by difference frequency mixing the signal and

idler beams into a type II AgGaS2, cut at θ=50.4°. The signal and idler beams are 2 mm dia.

before focusing with f=30 cm spherical mirrors. The generated mid-IR beam is collimated with

a spherical gold mirror. The residual signal and idler are removed by a 1-mm-thick Ge filter,

which is also used to overlap a collinear HeNe alignment beam on top of the mid-IR beam path.

We optimized the OPA configuration to generate the maximum bandwidth. Significant

amounts of dispersion occurs when pulses of <50 fs duration pass through even a 1-mm thick

material. Therefore, the thicknesses of the non-linear crystals must be optimized for bandwidth

and power. Fig. 2.5 shows how the mid-infrared spectrum depends upon the components

constituting our OPA. Starting with a 4 mm BBO and a 1.5 mm AgGaS2 crystal, which are

typical thicknesses in mid-IR OPAs (9), the OPA produced mid-IR pulses with a bandwidth of

200 nm and ~4 µJ of energy (Fig. 2.5, solid thick). Since the cube polarizer (14 mm thick) that

controls the white light generation stretches the 800 nm pulses from 50 to 77 fs, we began by

removing the cube polarizer. This change increased the bandwidth to ~300 nm (Fig. 2.5, dashed)
 30

without altering the energy of the mid-IR (the white light continuum was instead optimized by

translating the sapphire crystal along the beam focus). Turning to the crystals, we changed the

1.5 mm to a 1.0 mm thick AgGaS2 crystal, which brought the bandwidth to 400 nm, again

without power reduction (Fig. 2.5, solid thin). Replacing a 4 mm-thick BBO with a 2 mm BBO

(Fig. 2.5, dotted) did not alter the mid-IR spectrum, but the shot-to-shot stability of the mid-IR

improved and the power increased (~6 µJ). The shot-to-shot stability improved because the

signal and idler were no longer spontaneously generated without the white light seed present.

Finally, a 0.5 mm AgGaS2 dramatically increased the bandwidth to 850 nm (Fig. 2.5, dash-dot-

dashed), with only a slight sacrifice of energy (~5 µJ). Thus, the largest improvements are made

by proper choice of AgGaS2 crystals, which is consistent with the phase-matching bandwidths of

AgGaS2 increasing inversely to crystal thickness (570, 850 and 1700 nm for crystals with

thicknesses of 1.5, 1.0, and 0.5 mm, respectively). In the end, with proper selection of crystals

and optics, the bandwidth could be nearly quadrupled without significant loss of intensity,

corresponding to pulse durations as short as 43 fs if compressed.

 31

Figure 2.4 Layout of the home-built optical parametric amplifier based on β-barium borate

(BBO) crystal followed by difference frequency mixing with AgGaS2.

Regenerative SM AgGaS 2
MD type II
Amplifier f=30cm
SM 0.5mm
800 nm 1450 Au f=15cm
f=30cm θ=50.4 o
45 fs LWP
SM
1kHz Au
1mJ Ge filter
HR HR

BS Au mid-IR
R=90% Au Au f=25cm L4 HR
MD
HR MD
Cube
λ/2 Pol HR MD
BS L5
R=2% Au MD MD
f=-5cm
MD

HR HR Au 800 BBO 800

L3 L1 Sapp L2 SM
Au LWP type II LWP f=25cm
f=75cm f=10cm f=3cm
2mm
θ=25.9 o

Optics

BS beamsplitter HR high-reflecting mirror

L lens Au flat gold mirror

LWP long wave pass SM spherical gold mirror

MD manual delay
 32

Figure 2.5 Dependence of mid-IR bandwith on OPA optical configuration. (solid thick) 4

mm BBO and 1.5 mm AgGaS2; (dashed) cube polarizer is removed; (solid thin) 1.0 mm

AgGaS2; (dotted) 2 mm BBO; (dash-dot-dashed) 0.5 mm AgGaS2. The abrupt drop in intensity

at 4.3 µm is caused by CO2 absorption.

normalized intensity

4000 4200 4400 4600 4800 5000 5200 5400 5600 5800
wavelength (nm)
 33

2.4 Acknowledgments

We thank Professor David Blank and Dr. David Underwood for providing the design of the

home-built Ti:Sapphire oscillator.

2.5 References

1. Mukamel S (1995) Principles of onlinear Spectroscopy (Oxford University Press, New

York).

2. Asaki MT, Huang CP, Garvey D, Zhou JP, Kapteyn HC, & Murnane MM (1993)
Generation of 11-fs pulses from a self-mode-locked Ti-Sapphire laser. Opt. Lett.
18(12):977-979.

3. Backus S, Durfee CG, Murnane MM, & Kapteyn HC (1998) High power ultrafast lasers.
Rev. Sci. Instrum. 69(3):1207-1223.

4. Boyd RW (2003) onlinear Optics (Academic Press, New York) 2nd ed. Ed.

5. Dmitriev VG, Gurzadyan GG, & Nikogosyan DN (1999) Handbook of onlinear Optical
Crystals (Springer, New York) 3rd ed. Ed.

6. Siegman AE (1986) Lasers (University Science Books, Sausalito, CA) 1st ed. Ed.

7. Hamm P, Lauterwasser C, & Zinth W (1993) Generation of Tunable Subpicosecond

Light-Pulses in the Midinfrared between 4.5 and 11.5 Mu-M. Opt. Lett. 18(22):1943-
1945.

8. Kaindl RA, Wurm M, Reimann K, Hamm P, Weiner AM, & Woerner M (2000)
Generation, shaping, and characterization of intense femtosecond pulses tunable from 3
to 20 mu m. J. Opt. Soc. Am. B 17(12):2086-2094.

9. Hamm P, Kaindl RA, & Stenger J (2000) Noise suppression in femtosecond mid-infrared
light sources. Opt. Lett. 25(24):1798-1800.
 34

Chapter 3
Femtosecond Pulse Shaping Directly in the Mid-Infrared
Using Acousto-Optic Modulation

3.1 Introduction

Two-dimensional infrared (2D IR) spectroscopy is becoming an increasingly important tool for

studying the structures, environments and dynamics of molecules (1, 2). Collecting 2D or higher

dimensional spectra requires sequences of femtosecond and/or picosecond mid-IR pulses whose

time delays and phases can be specified. Currently, these pulse trains are generated using

beamsplitters where the delays are controlled with translation stages (3-5). This conventional

approach is sufficient to generate simple 2D IR spectra, but difficult to extend to higher

dimensions or to better extract desired quantities (6). Extending 2D IR requires more

sophisticated control over the pulse frequencies and phases. For example, recent simulations

have shown that the 2D IR cross peaks can be enhanced using pulse trains composed of pulses

with sophisticated chirps (7). Another example is work done in the near-IR, where 2D electronic

spectra were collected by cycling the phases of the pulses rather than using phase matching, in

analogy to the way that 2D NMR spectra are often recorded (8). Thus, mid-IR pulse shaping

promises to advance these new multidimensional infrared spectroscopies.

In the visible and near-IR spectral regions it is very common to use pulse shaping to

create trains of pulses or pulses with complicated chirps. These wavelength regions access

excited electronic states and can be used to optimize processes such as fluorescence, ionization

and photodissociation (9-11). Most experiments have utilized these wavelength regions the most
 35

because there exist a number of commercially available devices for shaping visible and near-

IR light, including liquid crystal modulators (12, 13), TeO2 acoustooptic modulators (14), and

deformable mirrors (15). However, these devices do not work in the mid-IR. Liquid crystal

modulators and TeO2 absorb wavelengths longer than 1.5 microns while deformable mirrors

have insufficient translation for a π-phase shift beyond λ=900 nm. Shaped mid-IR pulses have

been created indirectly, by shaping in the near-IR using a commercial shaper and transferring

that shape to the mid-IR via difference frequency mixing (16-19). However, the nonlinearity of

difference frequency mixing results in poor resolution and mid-IR intensities that strongly

depend on the pulse shape and phase. As a result, indirect shaping methods have limited utility

in 2D IR spectroscopy. A better way is to shape directly in the mid-IR. In this manner, intense

mid-IR pulses can first be generated from transform limited near-IR pulses and then shaped with

a resolution and efficiency dictated only by the design of the pulse shaper. Based on

groundbreaking work in TeO2 acousto-optic pulse shaping by Warren and co-workers (14), our

shaper uses germanium acousto-optic modulator (Ge AOM) directly modulates the phase and

amplitude of femtosecond mid-IR pulses, providing superior resolution and efficiency (20).

In this chapter, we report a pulse shaper built from a Ge AOM that works directly in the

mid-IR with a resolution and efficiency on par with visible pulse shapers. The high resolution

and efficiency enable our mid-IR shaper to be used for coherent control over ground states (21).

Unlike mid-IR generation via DFM, the mid-IR shaped by our Ge AOM is phase stable with

respect to the input mid-IR. As a result, these shapers can be used in experiments requiring high

phase stability such as 2D-IR spectroscopy.

 36

3.1.1 General theory of femtosecond pulse shaping

Femtosecond pulse shapers modulate the amplitude, the phase, or the polarization of a light pulse

in femtosecond duration. The electric field of a light pulse propagates at position r in time t in a

generalized form of

E(r, t ) = yˆ E (r, t ) = yˆ E0 exp[i (ω0t + φ − k ⋅ r )] , (3.1)

where ŷ is the unit vector defining the direction of E; E0 is the amplitude; ω0 is the center

frequency; φ is the phase; k is the wave vector and orthogonal to ŷ . A femtosecond pulse

shapers tailors E0, φ,  ŷ or a combination of the three. Polarization shapers, shaping are

rather new and most commercial pulse shapers control the amplitude and/or the phase.

Therefore, I will assume ŷ constant from now on.

In practice, most of electronic devices cannot shape light pulses in femtoseconds directly

in the time domain. Therefore, the incident electric field Ein (t ) is Fourier-transformed to give

~
Ein (ω ) , then modulated by a mask M (ω ) in frequency domain (22). This results in an outgoing

~
shaped spectral electric field Eout (ω ) :

~ ~
Eout (ω ) = Ein (ω )M (ω ) . (3.2)

~
The mask may modulate the spectral amplitude A(ω ) and the phase φ (ω ) , i.e.,

~
M (ω ) = A(ω ) exp[iφ (ω )] . (3.3)

~
Finally, Eout (ω ) is inversely Fourier-transformed to yield Eout (t ) in the time domain.
 37

3.1.2 General aparatus for femtosecond pulse shaping

Fig. 3.1(a) shows a basic pulse shaping apparatus, which consists of a spatial modulator that

serves as a mask and an optical configuration that Fourier-transforms from the time domain into

the frequency domain and vice versa (22). The spatial modulator is placed at the “Fourier plane”

inside a “4-f geometry” composed of two pairs of a grating and a lens spaced by f, the focal

length of the lens. The first pair of a grating and a lens disperses the incident pulse into

frequency, thus into space. Each frequency component of the incident beam is focused at the

focal plane of the lens (Fourier plane) and spatially separated along one dimension. The spatial

modulator located at this Fourier plain manipulates the amplitude and/or phase of the spatially

dispersed frequency elements at a computer’s command. Then, the second pair of a lens and a

grating − a mirror image of the first pair − transforms the pulses back to the time domain.

For instance, let us consider a case of generating a pulse pair: one pulse is a replica of the

incident pulse and another pulse is a copy of the incident pulse shifted forward in time by τ. Any

function gets shifted in time by τ when its Fourier-transformed counterpart in frequency is

multiplied with exp(iωτ), then inverse Fourier-transformed. Thus, a mask function for a pulse

pair with one pulse at t = 0 and another pulse at t = τ can be written as

1 iωτ
M (ω ) = (e + 1) (3.4)
2

where 1 generates another at t = 0 and exp(iωτ) generates the pulse at t = τ. Eq. 3.4 can be

rearranged to

M (ω ) = cos(ωτ / 2)eiωτ / 2 . (3.5)

 38
~
Thus, combining an amplitude mask A(ω ) = cos(ωτ / 2) and a phase mask φ (ω ) = ωτ / 2

generate a pulse pair composed of a fixed pulse and a scanning pulse with a time delay of τ. Fig.

3.1(b) displays how a pulse shaper converts one pulse into two pulses. The first pair of a grating

and a lens Fourier-transforms a short femtosecond pulse (gray dotted) into frequency domain. At

the focal plane, the spectrum of the incident pulse (gray solid) is multiplied by a sigmoidal

amplitude mask (thick, solid) and a linear phase mask (thick, dashed). After the second pair of a

lens and a grating transfers the resultant spectrum with fringes into time domain, the shaped

beam becomes a pair of pulses whose time delay is set by the periods of fringes in the spectrum.
 39

Figure 3.1 (a) A schematic of a femtosecond pulse shaper. (b) An example how a pulse

shaper generates a pulse pair with a time delay of τ.

(a)

f f f f

grating lens mask lens grating

1/τ τ
(b)

t ω ω ω t
 40

3.2 Experimental

3.2.1 Optical Configuration

Our pulse shaper is built from a germanium acousto-optic modulator (Isomet) that works directly

in the mid-IR between 2 and 18 µm (23, 24). Our design is based largely on work by Warren and

co-workers who pioneered AOM-based pulse shaping in the visible (14, 25). An AOM is a

programmable transmission grating whose efficiency and phase are modulated by an acoustic

wave pass through the AOM crystal. Fig. 3.2 details the setup of our pulse shaper. It follows the

general layout shown in Fig. 3.1 with three variations: First, we used cylindrical mirrors (f = 125

mm) instead of lenses to minimize material dispersion and aberration and to focus in one

dimension along the aperture of the modulator; Second, the Ge AOM is oriented at the Bragg

angle of ~2º to allow the diffracted beam to go through the remainder of the 4-f setup; Third, the

beam paths are separated in vertical dimension instead of the horizontal dimension. Gratings are

placed in quasi-Littrow configuration, where the incoming and the outgoing beams are the same

in angles, but separated in heights.

To align the shaper, we utilized a collinear HeNe beam overlapped on top of the mid-IR

beam path. ~55 fs mid-IR pulses (1.2x transform limited) are generated by DFM of the signal

and idler beams of a BBO optical parametric amplifier (OPA) in a AgGaS2 crystal (26). The

residual signal and idler are removed by a 1-mm-thick Ge filter, which is also used to overlap a

collinear HeNe alignment beam on top of the mid-IR beam path. The quasi-Littrow

configuration allows easy adaptability to multiple light sources such as a HeNe and mid-IR (27).

Alignment procedures are detailed below.

 41

(1) Before inserting the Ge AOM, the 4-f geometry of the cylindrical mirrors and gratings

is initially set using three parallel propagating HeNe beams diffracted from the first

grating with orders of 7, 8 and 9. To construct the quasi-Littrow configuration, first

grating deflects the incoming beam downward and a folding mirror is placed in a lower

height to allow the incoming beam to travel above the folding mirror. Build the first 2-f

geometry for The 7, 8 and 9th orders to have collimated beam path and horizontally

parallel spot shape by fine-tuning the tilt of the grating, the tilt of the cylindrical mirror

and the distance between the grating and the cylindrical mirror. Then, the second 2-f

geometry is constructed as a mirror image of the first 2-f. The second cylindrical mirror

reflects upward for the beam to pass above the second folding mirror, and then the

grating parallelizes the downward beam direction. Again, the second 2-f geometry is

completed with the tilt and the translation of the second pair of the grating and the

cylindrical mirror so that the outgoing beam from the shaper is collimated and parallel to

the ground.

(2) Let the mid-IR pass through the shaper, check the resultant spectrum and compare it with

the incoming spectrum. Block a small portion of each optic, check if any portion of the

mid-IR is clipped off by the optic, and then attune the grating angles and the vertical

alignment accordingly.

(3) Immediately before placing the AOM, the HeNe beams are horizontally tilted against the

expected deflection of 2º by the first folding mirror. Place the AOM at the Fourier plane,

and then tilt the AOM to maximize the diffracted mid-IR power outside the shaper. The

diffracted beam can be distinguished from the transmitted beam by electronically

chopping the acoustic wave propagating the AOM, so that the repetition rate of the
 42

shaped beam is 0.5 kHz. The AOM is designed to operate with an acoustic wave at a

75 MHz center frequency and a 50 MHz bandwidth. The acoustic wave is generated by a

piezoelectric transducer that converts a radio-frequency (RF) waveform from a 300

Msample/sec arbitrary waveform generator equipped in a computer. The acoustic wave

propagates along the 5.5-cm length of the AOM at 5.5 mm/µs, giving a time aperture of

10 µm. The trigger and timing of the acoustic wave will be discussed later in Sec. 3.3.2.

(4) The output power of the shaper is further tuned up by tweaking the folding mirrors before

and after the AOM as well as putting a waveplate in the incoming beam and rotating for

the optimal efficiency of the gratings and the Ge AOM. The Ge AOM has a deflection

efficiency of ~80%, giving a theoretical throughput efficiency of 50% when used in

conjunction with two gratings of 80% efficiency. We achieved 30% efficiency because

of suboptimal gratings and folding mirrors (~90% efficiency).

(5) The 2º deflection by the Ge AOM adds a linear chirp to the pulse. The chirp is

compensated by translating the second grating while maximizing the second harmonic

signal (SHG) of the shaped beam through a 0.5-mm thick doubling AgGaS2 cyrstal

(θ=33º). The leftover linear chirp and higher-order chirps are further compensated by

adding chirps to the phase of the acoustic wave that maximizes the SHG.
 43

Figure 3.2 The experimental setup of a mid-IR pulse shaper using a Ge acousto-optic

modulator. The angles of the incident and the outgoing beams for the grating are almost

identical (quasi-Littrow configuration), but the beam paths are separated in height: The incoming

beam for the shaper travels above the folding mirror (dashed line); The grating deflects the beam

downward (dotted line); The cylindrical mirror compensate the vertical tilt to parallelize the

beam to the optical table (solid); After Ge AOM, the optics are configured to a mirror image of

the setup before the Ge AOM.

Cylindrical Cylindrical
Mirror Mirror
Ge
AOM

Folding Folding
Mirror Mirror

Arbitrary
Grating Waveform Grating
Generator

Optics

Grating 300 grooves/mm (Newport)

Cylindrical mirror gold-coated, f=125 mm (CVI, custom-coated cylindrical lens)

Folding mirror Aluminum plane mirror (Thorlabs)

Ge AOM Germanium acousto-optic modulator (Isomet)

 44

3.2.2 Triggering and timing

Since the acoustic wave appears static on the timescale of the ultrafast pulse traversing the Ge

AOM, the acoustic wave acts as a modulated grating, deflecting desired frequencies with

amplitude and phase specified by the acoustic wave. As a result, the phase of the shaped mid-IR

pulses is set by the phase of the acoustic wave. Therefore, for pulses with reproducible phase

from one laser shot to the next, it is imperative that the arbitrary waveform generator is

synchronized to the phase and the repetition rate of the laser.

Fig. 3.3 details the synchronizing scheme. A photodiode monitors the repetition rate of

the Ti:sapphire oscillator as a reference for all the electronics afterwards. A custom-made

divider circuit uses the 88 MHz reference to generate a 1 kHz trigger pulse for both the arbitrary

waveform generator and the amplifying Nd:YLF pump laser. A 300 MHz clock signal is also

generated from the 88 MHz reference wave using a phase-locked loop circuit (also custom-made)

that serves as an external clock for the arbitrary waveform generator. This electronic

configuration produces clock and trigger pulses synchronized to within 0.6 ns, which is

substantially smaller than the periods of the 75 MHz acoustic wave (13.3 ns) and the 300 MHz

clock (3.3 ns). A 0.6 ns electronic jitter results in the theoretical phase stability of λ/50. Note

that the phase fluctuation cannot be reduced less than λ/4 when using the 300MHz “internal”

clock of the arbitrary waveform generator, which is not synchronized to the laser.

To time the bursts of acoustic wave to the incoming mid-IR pulses at the Fourier plane, a

delay generator defers the RF waveform right before the piezoelectric transducer. In our setup,

the electronic controller for the regenerative amplifier spends less time in response to the trigger

than the arbitrary waveform generator does. As a result, the acoustic wave arrives to the AOM

after the light pulse pass through the AOM. So we set the delay slightly less than 1 ms so that
 45

the acoustic wave burst encounters the next laser shot. The delay is further attuned by using a

short waveform burst centered within the 10-µs time aperture. While checking the deflected

spectrum with a monochromator, the delay is adjusted to place the narrow spectrum at the center

frequency of the mid-IR.

 46

Figure 3.3 Triggering and timing scheme of the laser and the shaper. The basis of the

timing sequence is the oscillator. A photodiode monitors the pulse train from a Ti:Sapphire

oscillator running at 88 MHz. The 88 MHz reference is sent to a divider and a phase-locked loop

that provides 1 kHz trigger and 300 MHz clock, respectively, for the arbitrary waveform

generator. The arbitrary waveform generator produces a 75 MHz RF waveform that is

electronically chopped to run 0.5 kHz. The RF waveform is delayed to locate the 10-µs time

aperture on top of the mid-IR pulse at the AOM. The delayed RF waveform is transformed into

an acoustic wave controlling the deflection at the Ge AOM.

11.4 ns

Ti:Sapphire
88 MHz Oscillator

1 ms 1 ms

Nd:YLF Regenerative OPA

Pump Laser 1 kHz Amplifier 1 kHz DFG

1 ms

Divider
Circuit 1 kHz
Pulse AOM
Shaper

Trigger 75 MHz
13.3 ns
Arbitrary
3.3 ns Waveform
Generator Delay Piezoelectric
Phase-locked 2 ms Generator Transducer
Loop Circuit Clock 0.5 kHz
300 MHz
 47

3.2.3 Characterizing pulse shapes

Fig. 3.3 shows optical devices for characterizing the shape of the pulse. A monochromator

equipped with a MCT (mercury cadmium telluride) array measures the spectrum in frequency

domain, while an auto- and a cross-correlator retrieve the amplitude and phase of the pulse in

time domain. The monochromator utilizes a focal length of 150 mm, giving a resolution of 3.6

nm. When characterizing the spectrum, we usually use a single pixel out of 64 pixels in the

MCD array detector to avoid the pixel-to-pixel variation. The auto- and cross-correlators are

collinear interferometers whose time delays are controlled with computer-controlled translational

stages outfitted with ZnSe wedges that afford a 0.02 fs time resolution and a maximum delay of

12 ps. The autocorrelation signal is generated by second-harmonic generation (SHG) of the

shaped beam focused into a 0.5-mm thick doubling AgGaS2 crystal (θ=33º) by an off-axis

parabolic mirror (f=50.8mm). A short-pass filter with a cut-off wavelength of 3.5 µm removes

the un-doubled mid-IR at 5~6 µm, while allowing the doubled mid-IR at 2.5~3 µm to pass

through. In the crosscorrelator, the shaped pulse is heterodyned by a small portion (2~3%) of the

unshaped beam that was reflected by an uncoated CaF2 wedge.

 48

Figure 3.4 The experimental setup for characterizing the shaped pulse. A monochromator

measures the spectrum, while the autocorrelator or the crosscorrelator retrieve the amplitude and

the phase in time.

pulse shaper

mid-IR G
CaF2 CM

AOM AWG

CM
G
FM

FM
DET
FM CD
MCT
BS array
DET
crosscorrelator monochromator

MD
CD
BS
SF
DET

SHG autocorrelator

Optics

CaF2 uncoated CaF2 wedge CM cylindrical mirror

G grating AWG arbitrary waveform generator

AOM acousto-optic modulator FM flipper mirror (gold)

MD manual delay CD computerized delay with ZnSe wedges

BS beamsplitter DET single MCT (mercury cadmium telluride) detector

SHG Doubling crystal SF short-pass filter

 49

3.3 Performance of the mid-IR pulse shaper

2D IR spectroscopy or coherent control experiments necessitates intense and phase stable pulses

with accurate pulse shape. In this section, we demonstrate these capabilities by resolving

spectrally and auto-/cross-correlating the shaped mid-IR.

3.3.1 Resolution

Our shaper is based on Ge AOM, which serves as a programmable diffractive grating whose

phase and efficiency is controlled by shaping the phase and the amplitude of the acoustic wave

passing through the Ge crystal. A 75-MHz acoustic wave with a bandwidth of ∆f = 50 MHz

travels the 5.5-cm window of the Ge crystal at a speed of 5.5 mm/µs, giving the time aperture, ∆t

= 10 µs. Thus, for our design, ∆t · ∆f = 500, which indicates a maximum of 500 equivalent

resolvable elements across the aperture.

To measure the actual resolution, we generated a comb-shaped spectrum with the

narrowest width that the arbitrary waveform generator can afford. Shown Fig. 3.5(b) is a

spectrum collected for a shaped pulse generated by a series of 3.3 ns acoustic waves separated by

665 ns, shown in Fig. 3.5(a). Each wave deflects a particular frequency in the mid-IR pulse

spectrum. The resulting peaks each have a FWHM of 5 nm and are separated by 63 nm,

demonstrating the high resolution and contrast ratio of the pulse shaper. From the 5 nm FWHM,

we estimate the actual resolution to be 190 equivalent pixels, after taking into account

monochromator resolution (3.6 nm) and timing jitter (1 ns jitter creates 0.1 nm broadening). The

limiting factor for resolution is currently the ~250 µm spot size, which is more than twice as
 50

large as the optimal resolvable element (110 µm). Thus, expanding the beam prior to the

pulse shaper will be an easy means to proportionally improve the resolution.

We use the comb-shaped spectrum to correlate the spatial position on the AOM and the

spectral position in the mid-IR spectrum for accurate shaping. The arbitrary waveform generator

digitizes the 10-µs time aperture of the RF waveform with an array of 3008 samples. A comb-

shaped RF waveform with a series of peaks with a width of one sample at every 100 samples was

used to generate the calibration curve shown in Fig. 3.5(c). A single-peak spectrum is also

measured to center the RF waveform in the mid-IR spectrum. The peak frequencies in the

resultant spectrum are measured in THz and the calibration curve correlating sample indices to

mid-IR frequencies is fitted by a second-order polynomial. The fitting parameters are used when

digitizing a designed mask in optical frequencies to a discrete set of samples for the arbitrary

waveform generator.
 51

Figure 3.5 Resolution measurements. (a) The RF signal used to modulate the spectrum. (b)

The spectrum resulting from the modulated RF waveform of (a). (c) The calibration curve

correlating times at the RF waveform to frequencies of the mid-IR. The measured peak

frequencies in the comb-spectrum (circles) are fitted by a second-order polynomial (solid line).

(a)
amplitude

1 3ns 665ns

0
0 2 4 6 8 10
time (µs)
(b)
intensity (a.u.)

63nm

5nm

4400 4600 4800 5000 5200 5400

wavelength (nm)
62
(c)
61
frequency of mid-IR (THz)

60

59

58

57

56

55

54

53
600 800 1000 1200 1400 1600 1800 2000
time (sample index)
 52

3.3.2 Efficiency

The shaper efficiency is of paramount importance in achieving sufficient pulse energies to be of

use in 2D IR and coherent control experiments. The Ge AOM has a deflection efficiency of

~60%, giving a theoretical throughput efficiency of 40% when used in conjuction with two

gratings of 80% efficiency. We usually achieve 30% efficiency because of suboptimal optics.

Most likely the folding mirrors is the limiting factor since the aluminum coating has ~90%

reflection efficiency. The total power of our shaped pulses is typically ~1.5 µJ when starting

from an OPA output of 5 µJ at 5 micron, and ~0.8 µJ when starting from an OPA output of 3.5

µJ at 6 micron.

To accurately program the pulse shape, the efficiency needs to be correlated for the

amplitude and the position of the acoustic wave. Shown in Fig. 3.6(a) is the deflection

efficiency of AOM as a function of acoustic wave amplitude. The deflection efficiency scales

nearly linearly with acoustic wave amplitude up to 0.5 of the maximum voltage of the arbitrary

waveform generator. This linear correlation holds across the 55-mm aperture, exampled at 10, 20

and 30 mm. Shown in Fig. 3.6(b) is the AOM deflection efficiency as a function of position

along the aperture for an acoustic wave amplitude of 0.5. The maximum deflection efficiency is

~70% and drops to ~50% at 15-mm from the end of the aperture due to divergence of the

acoustic wave. When shaping the pulses, we include the aperture position and acoustic wave

amplitude dependent deflection efficiencies in the software to design the shaped pulses.
 53

Figure 3.6 Amplitude and position dependence on deflection efficiency. (a) Deflected

intensity at three positions across the aperture vs. acoustic wave amplitude. (b) Deflection

efficiency across the aperture for tan acoustic wave amplitude of 0.5.

(a) (b)

deflection efficiency (%)

75
deflection efficiency

1.0
70
0.8
normalized

65
0.6
60
0.4 at 10 mm 55
at 20 mm
0.2 at 30 mm 50

0.0 45
0 0.2 0.4 0.6 0.8 1 5 10 15 20 25 30 35 40
normalized position at the aperture (mm)
acoustic wave intensity
 54

3.3.3 Stability

In order for shaped pulses to be generally incorporated into a coherent 2D IR pulse sequence,

they must be phase stable since the 2D IR signal is collected interferometrically. Interferometric

stability is degraded by changes in pathlength that are usually caused by variations in mirror

pointing stability. These fluctuations can be actively compensated with stabilized beampaths or

passive correction. Besides pathlength drift, phase instability can be caused by the Ge AOM

electronics. Since the phase of the acoustic wave sets the phase of the mid-IR pulse, the acoustic

wave must be reliably triggered to within a fraction of the 75 MHz center frequency for each

laser pulse. A timing jitter of 6.7 ns would lead to randomly phased pulses. The arbitrary

waveform generator locates samples separated by 3.3 ns according to a 300 MHz clock (internal

or external). When using the internal clock unsynchronized to the laser, the arbitrary waveform

generator produces rf waveforms with a phase jitter of λ/4 due to the timing jitter of 3.3 ns. For

an external clock, a phase-lock circuit generates a 300 MHz clock signal synchronized to the

Ti:Sapphire oscillator within 0.6 ns.

Shown in Fig. 3.7 are plots of the phase of the shaped pulse relative to an unshaped

reference during data collection of 100 sec, quantified by with our crosscorrelator. The phase

was measured by setting the delay stage to a position where the crosscorrelation signal amplitude

is close to zero so that intensities are linearly relative to the phase. Each point in the plot of Fig.

3.7 is averaged over 5 laser shot. Notice that the phase fluctuation with the internal clock (Fig.

3.7(a)) is ~4 times larger that the phase jitter with the external clock (Fig. 3.7(b)). The standard

deviation over the 100 second experiment gives a phase stability of λ/27 when the external clock

used. When normalized for shot-to-shot noise, this is about half the phase stability of a standard

2D IR optical setup without a shaper. Since the period of the acoustic wave corresponds to 13.3
 55

ns, with 0.6 ns electronic jitter, we do not expect better than λ/50 phase stability. Electronic

jitter might be further reduced by locking to a higher harmonic of the Ti:Sapphire oscillator.

Phase drift on longer timescales can be measured and corrected passively.

 56

Figure 3.7 Phase stability measurements using (b) the internal clock and (c) the external

clock for the arbitrary waveform generator. 5 laser shots were averaged.

π/2
π/4
(a)

0
phase (radian)

−π/4
−π/2
π/4 (b)
0
−π/4
π/2
0 10 20 30 40 50 60 70 80 90 100
time (sec)
 57

3.4 Applications of the mid-IR pulse shaper

3.4.1 Chirp compensation

Mid-IR pulses generated from the OPA are not necessarily transform-limited. Bragg deflection at

the AOM also adds a significant amount of linear chirp to the pulse and the Ge AOM itself

causes material dispersion. Since the 4-f configuration is a grating-pair compressor (22), the

linear chirp can be reduced by translating the second grating in the shaper to maximize the SHG

of the shaped beam using a doubling AgGaS2 crystal. Following grating optimization, we use

the AOM to refine the linear chirp compensation as well as compensate for higher order phase

distortions in the pulses. In fact, in order to program pulses with well-defined shapes, it is

essential to first remove the phase distortions of the input pulses. The phase of the electric field

in the frequency domain can be extended as

1 1
φ (ω ) = φ0 + φ1 (ω − ω 0 ) + φ2 (ω − ω 0 ) 2 + φ3 (ω − ω 0 ) 3 + ⋅ ⋅ ⋅ (3.6)
2 6

where φn are the dispersion coefficients, each of which can be independently varied.

To create transform limited pulses, we varied the 2nd and 3rd-order dispersion

coefficients (φ2 and φ3) while recording the resultant SHG signal, shown in Fig. 3.8. The

optimum values resulting in the maximum SHG were φ2 = 0.06 ps2 and φ3= 0.06 ps3, which

corresponds to the shortest pulse duration. With these optimum coefficients, the duration

measured from the autocorrelation in Fig. 3.8(b) was reduced to 67 fs from 80 fs. While this

process produces transform limited pulses, the pulse duration is not as short as might be expected

from the bandwidth of the pulses emitted directly from the OPA (Fig. 2) because the deflection

efficiency of the shaper effectively reduces the pulse bandwidth (Section 3.3.2, above).
 58

Figure 3.8 Compressing the shaped. (a) Scanned SHG intensity as a function of phase

terms φ2 and φ3. Autocorrelations with (b) unoptimized and (c) optimized phase parameters.

0.16
(a) (b) (c)
0.12
φ3 (ps3)

0.08

0.04

0.00

-0.04
-0.05 0.00 0.05 1.00 1.50 2.00 -200 -100 0 100 200 -200 -100 0 100 200
φ2 (ps2) time (fs) time (fs)
 59

3.4.2 Pulse pair generation

Nonlinear spectroscopies, such as 2D IR spectroscopy, rely on trains of pulses with controllable

phases and time delays. These pulse trains are conventionally created using beam splitters.

Pulse shaping has the potential of generating arbitrary numbers of pulses with control over the

amplitude and phase of each pulse, which is impossible for conventional splitting configurations.

Here, we create a pulse pair with variable time delay and phase. The mathematical

representation of generating a pulse pair in Eq. 3.4 can be extended to specify a phase for each

pulse in a pulse pair:

1 iωτ iφ1
M (ω ) = (e e + eiφ 2 ) (3.7)
2

where φ1 is the phase of the pulse at t = τ where as φ2 is the phase of the pulse at t = 0. The mask

function above can be rearranged as

 ωτ + φ1 − φ2   ωτ + φ1 + φ2 
M (ω ) = cos  exp i . (3.8)
 2   2 

Thus, an amplitude mask of cos[(ωτ + φ1 − φ2 ) / 2] combined with a phase mask of

(ωτ + φ1 + φ2 ) / 2 generates two pulses: one at t = τ with a phase of φ1; another at t = 0 with a

phase of φ2.

Fig. 3.9(a) includes the autocorrelation of a pulse pair created by sinusoidally modulating

the intensity of an RF waveform. The relative delay is determined by the period of the sine wave

while the phase of the sinusoidal modulation dictates the relative phase between the two pulses.

Also shown in Fig. 3.9(b) are enlarged sections of 1 ps autocorrelations with the relative phase

set to ∆φ = 0 and π, revealing a corresponding phase difference. In Fig. 3.9(c) the relative phase
 60

is incremented in steps of 0.1π rad, and the measured deviation from linearity gives a relative

phase control of the order of 0.008π rad. Accurate relative phase control is critical in phase

cycling 2D IR experiments.
 61

Figure 3.9 Pulse pairs with various spacing and relative phase. (a) The autocorrelation of

pulse pairs with time separations of 1, 2, and 3 ps. (b) Enlarged section of the 1 ps

autocorrelation from (a) with relative phases between pulses of 0 rad (solid) and π rad (dashed).

(c) Theoretical and experimental relative phases of pulse pairs with 1 ps time separation, for

which the relative phase has been varied in steps of 0.1π.

(a)
intensity (a.u.)

-3 -2 -1 0 1 2 3
time (ps)

(b) π (c)
experimental ∆φ
intensity (a.u.)

0 π/2

-1
0
960 1000 1040 1080 0 π/2 π
time (fs) theoretical ∆φ
 62

3.5. Conclusion

Mid-IR pulse shaping has been previously limited to simple linear chirps using material or

gratings and to indirect shaping methods with low resolution and power (16-19). Although

simply shaped, applications of these pulses demonstrated vibrational ladder climbing and

optimized pulse compression (28, 29). We demonstrated pulse shaping directly in the mid-IR

using a Ge AOM with high efficiency and resolution. We generated a series of phase and

amplitude pulses, the accuracy of which is tested with simulations. Phase-stable mid-IR shaping

could be used in multidimensional IR spectroscopies to map solution phase potentials, monitor

energy transfer, and study solute-solvent interactions. In fact, simulation studies suggest that

well-designed pulses can populate certain high vibrational modes (30), control intra- and inter-

molecular proton transfer (31-33), control electron transfer through vibrational excitation (34),

and direct isomerization (35). Considering that 2D IR experiments are usually carried out with

<1 µJ of power (5) and that vibrational populations have recently been inverted with only 2.2 µJ

(36), it stands to reason that this new shaper promises to open a new class of experiments geared

to understanding and optimizing ground state control.

 63

3.6. References

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U.S.A. 104(36):14189-14189.

2. Cho MH (2008) Coherent two-dimensional optical spectroscopy. Chem. Rev.

108(4):1331-1418.

3. Cowan ML, Bruner BD, Huse N, Dwyer JR, Chugh B, Nibbering ETJ, Elsaesser T, &
Miller RJD (2005) Ultrafast memory loss and energy redistribution in the hydrogen bond
network of liquid H2O. ature 434(7030):199-202.

4. Volkov V, Schanz R, & Hamm P (2005) Active phase stabilization in Fourier-transform

two-dimensional infrared spectroscopy. Opt. Lett. 30(15):2010-2012.

5. Ding F, Mukherjee P, & Zanni M (2006) Passively correcting phase drift in 2D IR

spectroscopy. Opt. Lett. 31(19):2918-2920.

6. Ding F & Zanni MT (2007) Heterodyned 3D IR spectroscopy. Chem. Phys. 341(1-3):95-

105.

7. Abramavicius D & Mukamel S (2004) Disentangling multidimensional femtosecond

spectra of excitons by pulse shaping with coherent control. J. Chem. Phys. 120(18):8373-
8378.

8. Tian PF, Keusters D, Suzaki Y, & Warren WS (2003) Femtosecond phase-coherent two-
dimensional spectroscopy. Science 300(5625):1553-1555.

9. Gordon RJ & Rice SA (1997) Active control of the dynamics of atoms and molecules.
Annu. Rev. Phys. Chem. 48:601-641.

10. Assion A, Baumert T, Bergt M, Brixner T, Kiefer B, Seyfried V, Strehle M, & Gerber G
(1998) Control of chemical reactions by feedback-optimized phase-shaped femtosecond
laser pulses. Science 282(5390):919-922.

11. Rabitz H, de Vivie-Riedle R, Motzkus M, & Kompa K (2000) Chemistry - Whither the
future of controlling quantum phenomena. Science 288(5467):824-828.

12. Weiner AM, Leaird DE, Patel JS, & Wullert JR (1990) Programmable Femtosecond
Pulse Shaping by Use of a Multielement Liquid-Crystal Phase Modulator. Opt. Lett.
15(6):326-328.

13. Wefers MM & Nelson KA (1993) Programmable Phase and Amplitude Femtosecond
Pulse Shaping. Opt. Lett. 18(23):2032-2034.

14. Dugan MA, Tull JX, & Warren WS (1997) High-resolution acousto-optic shaping of
unamplified and amplified femtosecond laser pulses. J. Opt. Soc. Am. B 14(9):2348-2358.
 64

15. Hacker M, Stobrawa G, Sauerbrey R, Buckup T, Motzkus M, Wildenhain M, &

Gehner A (2003) Micromirror SLM for femtosecond pulse shaping in the ultraviolet.
Appl. Phys. B 76(6):711-714.

16. Eickemeyer F, Kaindl RA, Woerner M, Elsaesser T, & Weiner AM (2000) Controlled
shaping of ultrafast electric field transients in the mid-infrared spectral range. Opt. Lett.
25(19):1472-1474.

17. Belabas N, Likforman JP, Canioni L, Bousquet B, & Joffre M (2001) Coherent
broadband pulse shaping in the mid infrared. Opt. Lett. 26(10):743-745.

18. Witte T, Zeidler D, Proch D, Kompa KL, & Motzkus M (2002) Programmable
amplitude- and phase-modulated femtosecond laser pulses in the mid-infrared. Opt. Lett.
27(2):131-133.

19. Tan HS & Warren WS (2003) Mid infrared pulse shaping by optical parametric
amplification and its application to optical free induction decay measurement. Opt.
Express 11(9):1021-1028.

20. Warren WS (2006) In unpublished work, W. S. Warren investigated picosecond mid-IR

pulse shaping using a Ge AOM.).

21. Strasfeld DB, Shim S-H, & Zanni MT (2007) Controlling vibrational excitation with
shaped mid-IR pulses. Phys. Rev. Lett. 99(3).

22. Weiner AM (2000) Femtosecond pulse shaping using spatial light modulators. Rev. Sci.
Instrum. 71(5):1929-1960.

23. Shim S-H, Strasfeld DB, Fulmer EC, & Zanni MT (2006) Femtosecond pulse shaping
directly in the mid-IR using acousto-optic modulation. Opt. Lett. 31(6):838-840.

24. Shim S-H, Strasfeld DB, & Zanni MT (2006) Generation and characterization of phase
and amplitude shaped femtosecond mid-IR pulses. Opt. Express 14(26):13120-13130.

25. Hillegas CW, Tull JX, Goswami D, Strickland D, & Warren WS (1994) Femtosecond
Laser-Pulse Shaping by Use of Microsecond Radiofrequency Pulses. Opt. Lett.
19(10):737-739.

26. Kaindl RA, Wurm M, Reimann K, Hamm P, Weiner AM, & Woerner M (2000)
Generation, shaping, and characterization of intense femtosecond pulses tunable from 3
to 20 mu m. J. Opt. Soc. Am. B 17(12):2086-2094.

27. Prakelt A, Wollenhaupt M, Assion A, Horn C, Sarpe-Tudoran C, Winter M, & Baumert T

(2003) Compact, robust, and flexible setup for femtosecond pulse shaping. Rev. Sci.
Instrum. 74(11):4950-4953.

28. Kleiman VD, Arrivo SM, Melinger JS, & Heilweil EJ (1998) Controlling condensed-
phase vibrational excitation with tailored infrared pulses. Chem. Phys. 233(2-3):207-216.
 65

29. Demirdoven N, Khalil M, Golonzka O, & Tokmakoff A (2002) Dispersion

compensation with optical materials for compression of intense sub-100-fs mid-infrared
pulses. 27(6):433-435.

30. Beyvers S, Ohtsuki Y, & Saalfrank P (2006) Optimal control in a dissipative system:
Vibrational excitation of CO/Cu(100) by IR pulses. J. Chem. Phys. 124(23):234706.

31. Doslic N, Sundermann K, Gonzalez L, Mo O, Giraud-Girard J, & Kuhn O (1999)

Ultrafast photoinduced dissipative hydrogen switching dynamics in thioacetylacetone.
Phys. Chem. Chem. Phys. 1(6):1249-1257.

32. Ohta Y, Bando T, Yoshimoto T, Nishi K, Nagao H, & Nishikawa K (2001) Control of
intramolecular proton transfer by a laser field. J. Phys. Chem. A 105(34):8031-8037.

33. Petkovic M & Kuhn O (2004) Ultrafast wave packet dynamics of an intramolecular
hydrogen transfer system: from vibrational motion to reaction control. Chem. Phys.
304(1-2):91-102.

34. Skourtis SS, Waldeck DH, & Beratan DN (2004) Inelastic electron tunneling erases
coupling-pathway interferences. J. Phys. Chem. B 108(40):15511-15518.

35. Artamonov M, Ho TS, & Rabitz H (2006) Quantum optimal control of molecular
isomerization in the presence of a competing dissociation channel. J. Chem. Phys. 124(6).

36. Ventalon C, Fraser JM, Vos MH, Alexandrou A, Martin JL, & Joffre M (2004) Coherent
vibrational climbing in carboxyhemoglobin. Proc. atl. Acad. Sci. U.S.A. 101(36):13216-
13220.
 66

Chapter 4
Automating 2D IR Spectroscopy Using Mid-Infrared Pulse
Shaping

4.1 Introduction

Two-dimensional infrared (2D IR) spectroscopy is becoming a very useful tool for probing the

fast structural dynamics of chemical and biological systems (1, 2). Analogous in many ways to

2D NMR spectroscopy, 2D IR spectroscopy probes molecular structures through vibrational

frequencies, couplings, and transition dipole angles (3-7). Environmental dynamics are probed

through lineshape analysis (8-10). The combination of structural sensitivity and fast time-

resolution (fs/ps) makes this technique and its variants (11-14) especially adept at monitoring

dynamics of evolving structures or the kinetics of chemical reactions (15-18). Although it is a

powerful technique, implementing 2D IR spectroscopy is technically challenging. In a typical

2D IR spectrometer, each pulse has its own optical path composed of several mirrors to route the

beam to the sample and a mechanical delay stage that controls the time delay by changing the

physical length of the optical path. All of the pulses have the same frequencies and shapes

unless additional optics are added such as an etalon to narrow a pulse bandwidth (3, 19) or a

second mixing crystal to generate pulse sequences with two center frequencies (20, 21). Thus,

implementing even the simplest pulse sequence is a tremendous amount of work. This is in

sharp contrast to 2D NMR spectroscopy, where NMR pulse sequences are easily programmed

with precisely set frequencies, time-delays, phases and intensities. As a result, it is

straightforward in NMR to change the pulse sequences or add additional pulses in order to create
 67

the best spectrum for measuring the desired information. In this chapter, we take a step closer

towards making 2D IR more like 2D NMR spectroscopy. Using a mid-IR pulse shaper recently

developed by our group (see Chapter 3), we can now programmably generate 2D IR pulse

sequences with controlled phases, delays and shapes (22, 23). Using traditional 2D IR methods

as a starting point, we improve upon their accuracy, speed and ease of implementation with

several alternative pulse sequences optimized to maximize the structural content of the spectra

(24). We also introduce phase control and rapid-scan data collection to tackle practical issues in

applying the method to biological systems evolving continuously.

This chapter is focused on 2D IR spectroscopy, but in collaboration with Niels Damrauer

and his group at the University of Colorado-Boulder, we also recently extended our pulse

shaping approach to 2D visible (2D Vis) spectroscopies (25) which are being used to explore the

electronic couplings and energy transfer processes in photo-biological systems and

semiconductors. While 2D IR and 2D Vis spectroscopies probe very different processes, their

implementation is virtually identical. The only important difference is that the wavelength is

much shorter in the visible, which puts a stringent requirement on the phase stability of 2D Vis

spectrometers. However, this tricky problem is automatically overcome with pulse shaping. In

this chapter, we focus on 2D IR spectroscopy, but because the infrared and visible techniques are

so similar, the contents for 2D IR can also be used to understand pulse shaping methods for

collecting 2D Vis spectroscopies as well. To aid in the comparison, a section on 2D Vis

spectroscopy is also included to outline salient differences.

 68

4.1.1 Conventional methods of 2D IR spectroscopy

The most obvious way of collecting 2D IR spectra would be stacking pump-probe spectra from

narrow-band pump pulses whose center frequencies are scanned. This so-called “hole-burning”

method was indeed the first method of collecting 2D IR spectra introduced by Hamm, Lim and

Hochstrasser (3) using etalons. An etalon consists of two partial reflectors aligned parallel so

that a pulse entering on one end bounces back and force between the two mirrors, leaking a

portion of the pulse with each bounce. The leaked portions interfere with one another, creating a

narrow-band whose center frequency is determined by the distance between the two mirrors.

The output pulse shape in time contains a long exponential tail as in Fig. 4.1(a), which limits the

time delay between the pump and the probe, thus limits time-resolution of the measurement to >1

ps. Moreover, the lineshape along the pump axis broadens due to the convolution of the intrinsic

linewidth of the vibrator with the bandwidth of the pump pulse.

In 2000, Hochstrasser and coworkers implemented a pulsed version of 2D IR

spectroscopy that is analogous in many ways to modern pulsed 2D NMR spectroscopy (Fig. 1b)

(8, 11). Hinted from Fourier-transform (FT) spectroscopy useful for improving time and

frequency resolution, both the pump and the probe beams are split into two transform-limited

pulses whose time delays are scanned and Fourier-transformed to generate two frequency axes.

In practice, the first three pulses serves as excitation pulses aligned in a boxcar geometry (Fig.

4.1(b)). The excitation beams are separated in space by beamsplitters and separated in time by

translational stages. A signal comes off at a direction determined by a vector sum of the three

beams. A local oscillator pulse is collinearly combined to the signal for sampling the amplitude

and the phase of the emitted signal. Best time resolution is achieved by using transform-limited

pump pulses; Best frequency resolution is obtained by using proper time increments and number
 69

of data. Moreover, this method provides superior selectivity of choosing a specific signal by

spreading combinations of excitations in space according to their phase matching conditions and

by controlling polarizations of all four pulses to selectively allow a combination of transitions at

a specific order of angles. However, it is challenging to separate and re-overlap the four pulses

using many numbers of optics and translational stages while keeping the phase stability of all

four beams within a small fraction of a micrometer. Moreover, the method does not

automatically generate absorptive spectra or spectra that are properly phased, unlike its hole-

burning counterpart. Instead, two separate 2D IR spectra must be collected with different pulse

sequences and added together to generate absorptive features and optimal spectral resolution (26).

Performing this addition is tricky and requires an appropriate pump-probe spectrum (27), which

does not exist for all pulse sequences. In addition, data collection is usually slow because each

time delay accompanies hundreds of millisecond of a wait time for a computer to communicate

with translational stages. Thus, any means that is more robust and rapid to generate and control

pulse sequence would greatly improve the methods for collecting 2D IR spectra.

A mid-IR pulse shaper is a convenient tool to generate pulse sequences with accurate and

stable phase (22, 23). Femtosecond pulse shaping allows arbitrary control over amplitude and/or

phase and is often capable to generate very complicated shapes that are almost impossible for

other optical means to reproduce (28). Generating a sequence of pulses is one of the simplest

applications to demonstrate performance of pulse shapers. Therefore, most of available pulse

shapers are ready to carry out 2D optical spectroscopy including our mid-IR pulse shaper. Our

shaper is based on Ge AOM, which serves as a programmable diffractive grating whose phase

and efficiency is controlled by shaping the phase and the amplitude of the acoustic wave passing

through the Ge crystal. Our design affords a maximum of 500 equivalent resolvable elements
 70

across the aperture. The actual resolution is 190 equivalent pixels, which enables us to

generate a pulse pair with a time delay as large as 13 ps (22). Our shaper is also demonstrated to

generate pulse pairs with relative phases within an accuracy of 0.008π rad. Also the relative

phases are perfectly stable during hours of data collection because the two pump pulses pass

through exactly the same optical setup. Even their absolute phases were demonstrated to be

stable within λ/27 over 100 seconds for 5 micron light because there is no moving part during

data collection (23).

4.1.2 Principles of generating 2D IR spectrum in a pump-probe geometry

Regardless of choice of method, generating 2D IR spectra rely on the same principles. Three

interactions with a light pulse are required for the sample to generate a 3rd-order nonlinear signal.

When a pulse interact with the sample just once, at least three pulses are necessary. In some

cases like hole-burning, the same pulse interacts with sample twice and generate 2D IR signal.

When a pump-probe geometry is used, the probe serves as a one of the three interactions as well

as a local oscillator that heterodynes the emitted field. The measured 2D IR signal can be written

as convolution of the response functions with the electric fields of the input pulses;

S (τ , T , ω probe ) = ∑ ∫ (Rn (τ , T , t ) ⊗ E1 ⊗ E 2 ⊗ E probe + E probe )e

−iω probet 2
dt (4.1)
n

where E1, E2, and Eprobe are the three interacting electric fields and each of them can be written as

Ei = Ai exp[i (ω0 t + φi − k i ⋅ r )] where A, ω0, φ, and k stands for the amplitude, the center

frequency, the phase and the wavevector, respectively, for i=1,2 and probe; Rn (τ , T , t ) are the

vibrational and orientational responses of the system; and ⊗ s represent convolutions of the
 71

electric fields with the response function. The electric field of a light, Ei, is convoluted with or

without complex conjugation depending on which response of the system interacts with the

electric field. Therefore, phase cycling and phase matching conditions are interchangeable with

each other.

When a hole-burning scheme is used, the shaper carves a narrow band that serves as both

E1 and E2, and scans the center frequency of the narrow band. A 2D spectrum is generated by

stacking pump-probe spectra along the center frequency of the narrow-band pump. When a pulse

pair is used instead of a narrow band, the time delay, τ, between the pulses is scanned and the

second frequency dimension is generated by Fourier-transfoming along τ ;

− iω pumpτ
S (ωpump , T , ωprobe ) = ∫ S (τ , T , ωprobe )e dτ . (4.2)

Consequently, the lineshapes of the 2D IR spectra depend upon the pulse shape used because the

linewidth along the pump axis is convolution of the intrinsic linewidth of the sample with the

pump shape.
 72

Figure 4.1 Schematic layouts of 2D IR experiments: (a) hole-burning method, (b) pulsed

photon echo method, and (c) partially collinear method.

(a) T
k1
k2 kS

k3

(b) t T τ
k1
kS
k2

k3
kLO

(c) T t
k1
k2 kS

k3
 73

4.2 Experimental

4.2.1 Pump-probe spectrometer

Shown Fig. 4.2(a) is the optical layout of the experimental setup for the automated 2D IR

spectroscopy. A mid-IR pulse at 2~18 micron and ~50 fs duration is generated by difference

frequency mixing the signal and the idler from a BBO-based optical parametric amplifier that is

pumped by a 45-fs Ti:sapphire regenerative amplifier running at 1 kHz. A small fraction of the

IR beam is served as a probe, and the remainder is sent to a mid-IR pulse shaper described in

Chapter 3 and serves as a pump. The pulse shape of the pump beam is tailored by a Ge AOM, a

modulated grating, whose phase and efficiency across the aperture is controlled by a 75-MHz

acoustic wave (22). An arbitrary waveform generator equipped on a computer produces an RF

waveform at 300 Msample/s, and then the resultant RF waveform is amplified and transferred to

the acoustic wave. A pair of parabolic mirrors focuses and collimates both the pump and the

probe at the sample. The pump and the probe are overlapped temporally at the sample using a

mechanical time delay placed at the pump pass while keeping the probe path length constant.

The probe beam is spectrally dispersed with a monochromator and its spectrum is measured with

a MCT (mercury cadmium telluride) array detector with 64 pixels. Changes of the probe

absorption are measured with and without presence of the pump pulses that are tailored to spread

the probe absorption along the pump axis. Since the shaper allows arbitrary pump shapes, the

setup can carry out both the hole-burning and the pulsed photon echo experiments. For the hole-

burning, the shaper slices a narrow band out of the input spectrum at various center frequencies

and the resultant probe absorption spectra are stacked along the frequency of the narrow-band

pump to give ωpump. For the pulsed photon echo, pulse pairs are generated with their time delay
 74

varied from 0 to a time delay long enough to allow vibrations relaxes completely. Then the

time scans at each ωprobe are Fourier-transformed to generate the second frequency domain of

ωpump.

4.2.2 Waveform generation

Shown in Fig 4.2(b) is the electronics layout to cycle a new shape for every laser shot and collect

data without deadtime. Data collection in this method is two to three orders of magnitude faster

than conventional methods because the setup uses no moving mechanics. In conventional

methods, one or two automatic stages are translated to a new position to scan the center

frequencies of the etalon output or the time delays between pulses. At each translation, the

computer spends hundreds of millisecond for communicating with the automatic stages. In

contrast, it only takes 10 µs for the Ge AOM to upload a new mask. Therefore, every laser shot

can bear a new shape since the repetition rate of the light source is usually 1~20 kHz.

To reduce the communication time between the computer and the arbitrary waveform

generator (CompuGen 4300, GaGe Applied Technologies), usually 426 waveforms are loaded to

the 1 M on-board memory of the arbitrary waveform generator at once prior to the experiment (it

takes ~40 seconds to upload 426 waveforms). A time aperture of 10 µs at Ge crystal is filled up

with 3008 samples when 300 MHz clock used. Then, 1 M memory can hold 340 waveforms

consist of 3008 samples. Usually, since the deflection efficiency diminishes as the acoustic wave

propagates on the crystal from the piezoelectric transducer, we use only 2400 samples close to

the transducer and load 426 waveforms to the 1M memory of the arbitrary waveform generator.
 75

The arbitrary waveform generator has four output channels and four 1M memory for each

channel.

Once the 426 waveforms are loaded, the arbitrary waveform generator brings out each

waveform from its own memory and cycles through the loaded waveforms, one at a time, at each

trigger of 1 kHz from the regenerative amplifier. Thus, at 1 kHz, an entire 2D IR data set is

collected in only 0.426 sec. This process is continuously repeated until the necessary signal to

noise is averaged. Each waveform is synchronized to the laser by a trigger and a clock signal

generated from a 88 MHz pulse train from a photodiode monitoring the Ti:Sapphire oscillator.

The 1 kHz trigger is generated from a circuit that divides the 88 MHz of the Ti:Sapphire

oscillator by 88,000 to produce a 1 kHz TTL pulse. The 300 MHz external clock is produced

from a phase-locked loop circuit that relates the clock to the frequency and phase of the 88 MHz

reference. With the trigger and clock synchronized to the 88 MHz reference, the RF waveform is

synchronized to the 88 MHz reference within 0.6 ns.

As the 426 RF waveforms cycles continuously, the resultant 2D IR signal also cycles

accordingly. To catch the initiation point of the 426 waveform set, a “start” reference signal in

TTL format is also generated from the arbitrary waveform generator. In addition, every two

waveforms within 426 waveforms are paired to generate one pump-probe signal, thus a

“chopper” reference is necessary to process the data correctly. These reference signals can be

loaded to the arbitrary waveform generator along with the 426 pump shapes since the arbitrary

waveform generator affords four output channels sharing the same trigger and clock. Four

separate sets of 426 waveforms can be uploaded to the four channels all at once and cycled

synchronously with each other. Channel 1~4 of the arbitrary waveform generator are used as

follows.
 76

(1) Channel 1 produces a “start” reference for the array integrator. It contains a square

pulse at the first element of the waveform set and a zero voltage elsewhere. Thus, for a

set containing 426 waveforms, the reference repeats every 426 ms, thus the repetition rate

of the reference becomes 2.35 Hz. This reference is sent to the integrator electronics box

for the 64-pixel MCT array detector and used to detect the starting point of the 426

signals. All the channels in the integrator shares one gate pulse that is set for the probe

beam impinging the array detector located meters away from the Ge AOM. Thus, the

signal from the arbitrary waveform generator that is timed for the Ge AOM arrives to the

integrator a few microseconds earlier than the gate pulse. To retard the reference closer

to the gate without wasting the memory of the arbitrary waveform generator, we use a

separate delay generator for the reference before sent to the array integrator.

(2) Channel 2 creates a chopper reference signal for the array integrator. In practice, to

reduce number of delay generators, Channel 2 is not utilized for data collection and the

“start” reference is used instead for chopping the signal at Channel 4 because the start

reference indirectly indicates the chopping order. However, in case of collecting the data

less than 426 ms (for instance, checking the pump-probe signal from a reference pump

shaped with the RF waveform from Channel 3), Channel 2 will be ideal to chop the

signal. Then, another delay generator would be necessary.

(3) Channel 3 generates a reference pump shape for the Ge AOM to generate a single

transform-limited pulse. The reference pump is turned on/off every other laser shot

electronically to generate a reference pump-probe signal, which we refer for optimizing

the optical alignment. Since either Channel 3 or Channel 4 should be sent to the Ge
 77

AOM, a two-way switch is used to allow the user to switch between aligning the setup

and collecting 2D IR spectra.

(4) Channel 4 is loaded with the 426 waveforms containing 426 different pump shapes

varying time delays of pulse pairs or center frequencies of narrow bands. The output of

Channel 4 is sent to the Ge AOM to scan time delays or center frequencies for a 2D IR

spectrum. Since pump-probe signals are calculated from pairs of probe intensities with

and without present of the pump, 426 RF waveforms correspond to 213 time delays or

center frequencies. Thus, our setup collects a single 2D IR spectrum with 213 scanning

parameters at maximum, limited by the memory of the arbitrary waveform generator. To

use a larger number of scanning parameters, an arbitrary waveform generator with a

larger memory than 1M for each channel is required.

4.2.3 Signal detection

The array integrator (IR-0128, Infrared Systems Development Corp.) collects data from 62

channels measuring spectrally resolved intensities of the probe beam as well as data from the

external input monitoring the reference indicating the start point of the waveform set. The array

integrator affords total 128 channels for inputs: Channel 1~62 for one 64-pixel array; Channel

65~126 for another 64-pixel array; Channel 63~64 for two external input channels for signals

with ±1V maximum from single detectors to be integrated; Channel 127~128 for two external

input channels of DC-10 kHz level sensor for integrated signals and TTL pulses in ±10V

maximum. The integrator also includes one sync input to generate a gate pulse synchronized to

the trigger read from the sync input. During data collection, Channel 65~126 collects the probe

spectrum from one of the 64-pixel arrays and Channel 127 detects the start reference. After
 78

integrating channel 1~126 and detecting channel 127~128, the data from all 128 channels are

sent to the computer. When 426 waveforms used, the computer collects 426 data from the

integrator. Since the computer randomly initiate data collection, the 426 data are not necessarily

ordered in a specific sequence of time delays or center frequencies. Thus, the computer use the

start reference to correlate each data to the shape of the pump pulse, i.e. the waveform sent to the

Ge AOM. The start reference is used to assign the initiation point (time zero when time delays

are scanned) of the 426 data as well as to pair the 426 data to calculate changes of optical density,

∆OD, for 213 time delays or center frequencies. Each pump-probe signal, ∆OD, is calculated as

I 
∆OD = − log on  (4.3)
 I off 

where Is are the spectrally resolved intensities of the probe beam when the pump is turned on/off.

Note that ∆OD is equivalent to the 2D IR signal, S(τ, T, ωprobe) in Eq. 4.1. A single 2D IR

spectrum is collected as 2D array of 62x213 ∆OD signals from 213 scanning parameters (time

delay or center frequency) and 62 pixels (probe frequency). ωprobe is optically resolved by the

monochromator to the 62 pixels, whereas ωpump is numerically given after the data collection is

completed. When hole-burning method in frequency domain is used, 213 pump-probe spectra

are stacked along the center frequency of the narrow-band pump to give ωpump. When pulsed

photon echo method in time domain is used, time scans composed of 213 time delays are

Fourier-transformed to give ωpump.

 79

Figure 4.2 Experimental setup with (a) optical and (b) electronical layouts.

(a) optics

unshaped probe
mid-IR light
5~6 µm,
~4 µJ/pulse Ge AOM
~55 fs duration shaped pump

sample

λ/2 P

mono- MCT
chromator array

(b) electronics
Ti:Sapphire
Oscillator
1 ms

Divider
88 MHz Circuit 1 kHz AWG
ch4
TRIG

switch
ch3 Sync IN
Array IN
Phase-locked ch2
CLK

Loop Circuit 300 MHz ch1 Delay Ext IN

Generator

OUT

Computer Integrator
 80

4.3 Frequency-domain method

Pump-probe geometries have been used for hole-burning experiments to generate 2D IR

spectrum (3, 29). To compare the automated 2D IR with conventional 2D IR methods, we used a

mid-IR pulse shaper as a tool to generate a pump pulse with a narrow bandwidth (22, 23). To

demonstrate the utility of collecting 2D IR spectra with our shaper, in what follows we show a

series of 2D IR spectra that have been collected with differently shaped pulse sequences (30).

We start by mimicking the conventional hole-burning approach and then sequentially optimize

the spectra with improved pulse sequences. Shown in Fig. 4 are four series of 2D IR spectra

collected for W(CO)6 solvated in n-hexane, which is a model compound that has a single IR

active anti-symmetric stretch near 5 microns. Each series is generated using a different pump

shape.

First choice among many potential shapes was an etalon-like shape that is used in the

conventional hole-burning method. An etalon consists of two partial reflectors aligned parallel so

that a pulse entering on one end bounces back and force between the two mirrors, leaking a

portion of the pulse with each bounce. The leaked portions interfere with one another, creating a

narrow-band whose center frequency is determined by the distance between the two mirrors, d,

according to

1− R
M (ω ) = (4.4)
1 − R ⋅ exp[+ i (2d cosθ / c)ω ]

where R is the reflection coefficient, θ is the angle the light travels through the etalon and c is the

speed of light (31). To generate the 2D IR spectra in the first column of Fig. 4.3, we set the

bandwidth to 4.6 cm-1 (which is slightly larger than the homogeneous linewidth of 4.4 cm-1),
 81

scanned d, and plotted the change in absorption of the probe with and without the pump. 2D

IR spectra were collected at two different pump-probe delay times, T = 2.0 and 0.2 ps. At T =

2.0 ps, a negative and a positive peak pair is observed (blue and red in Fig. 4.3, respectively),

which is exactly as expected according to standard hole burning approaches on metal carbonyl

systems. The negative peak appears along the diagonal with ωpump = ωprobe = 1983 cm-1, which is

the fundamental frequency of W(CO)6. The positive peak is shifted by ∆ωprobe = 13.3 cm-1,

which is the anharmonicity of the W(CO)6 antisymmetric stretch (32). The spectra contain all of

the desirable characteristics about hole-burning that we discussed above: it is properly phased

without any post-data corrections and the peaks have absorptive lineshapes. Thus, pulse shaping

can reproduce the data collected using an etalon.

A significant drawback of the hole burning approach is that the picosecond pump pulse

can distort the signal. An etalon generates a picosecond pulse with a sharp front and a long

exponential tail (Fig. 4.3(a)). As a result, the 2D IR spectra collected with an etalon-type pump,

when separated 2 ps from the probe, generates peaks elongated along ωpump because the natural

linewidth is convoluted with a Lorentzian spectrum of a 4.6-cm-1 bandwidth (Fig. 4.3(b)). When

the exponential tail of the etalon-shaped pump is close enough to overlap with the probe

significantly (T = 0.2 ps), the 2D IR peak shapes were severely distorted (Fig. 4.3(c)). This

probably was because the portion of the pump pulse coming after the probe served as the second

and the third pulse among the three excitations generating the non-rephasing 3rd-order signal and

interfered with the desired 2D IR signal. For this reason, an etalon-type shape can be used only

when T > 1 ps. This could be a significant drawback for biological applications. For instance,

the amide I band of a peptide or protein, for which the vibration lifetime is ~1.4 ps, a 3×

enhancement of the signal strength would occur for T = 0.2 vs. 1.8 ps. Of course, the tail can be
 82

minimized by using a shorter duration for the pump pulse, but this leads to a larger bandwidth

and poorer frequency resolution in the 2D IR spectra.

Pulse shaping provides alternative approaches for data collection using hole burning.

With pulse shaping, the time and frequency profiles of the pump pulse are not limited to just

what can be created by an etalon, but can be programmed to optimize the spectral features. For

instance, we devised a new kind of the etalon-like shape with the same frequency distribution,

but flipped the direction of the exponential tail in time so that it decays away from the probe

pulse (Fig.4.3(d)). This may be almost impossible to accomplish with a conventional optical

setup, but a pulse shaper can easily reverse the pulse shape by reversing the sign of the phase

term in Eq.4.4. This new shape of “time-reversed” etalon produced similar 2D IR peak shapes

for both T = 0.2 and 2.0 ps without peak distortion (Fig. 4.3(e) and (f)). Since the pump pulse

tails away from the probe, the pump and probe pulses do not overlap until much shorter T,

resulting in smaller peak distortions. Furthermore, the signal strength is also stronger for a time-

reversed etalon pulse shape, since the population has had less time to relax with shorter T.

Regardless of the direction of tail in time, the Lorentzian-shaped spectrum of etalon-like

shapes is disadvantageous for its long baseline in frequency. The 2D IR spectrum is a

convolution of the pump spectrum with the natural linewidth of the molecular vibration (Eq. 4.1).

Thus, the etalon lineshapes in Lorentzian have a very broad baseline, and as a result, the 2D

peaks have broad tails, which degrades the frequency resolution. An improvement would be to

use a pump shape that reaches baseline more quickly.

An alternative shape of a Gaussian with a 7.0-cm-1 FWHM generated peak shapes with a

shorter tail along ωpump (Fig. 4.3(g)). Note that the tails along pump axis are now much shorter,

which is because the Gaussians decay much more rapidly than Lorentzians and thus have
 83

improved resolution even for pump pulses with the same full-width-at-half-maximum (Fig.

4.3(h) and (i)). Although we have explored Lorentzians and Gaussians here, any number of

other frequency-domain shapes could be used. Nonetheless, as long as a narrow-band pump

used, the linewidth measured with such a pump suffers from elongation or distortion. If one tried

a pump with a narrower band to reduce the elongation effect, the pulse duration in time would

extend even further and make workable T even farther out. Therefore, using transform-limited

pulses would be an ideal solution. The peak shape distortion could be negligible when the

response function is convoluted with a pulse far shorter than the lifetime of the response function

so that the light pulse can be approximated as a delta function, an infinitely sharp pulse. A time-

domain method utilizing transform-limited pulses will be discussed in the next section.
 84

Figure 4.3 2D IR spectra of W(CO)6 dissolved in hexane, obtained with pump pulse

shapes: (a-c) a traditional etalon; (d-f) a time-reversed etalon; (g-i) a Gaussian; (j-l) a pulse pair.

The left column shows the pulse shapes in time domain, and the center and right columns are 2D

IR spectra for T = 2.0 and 0.2 ps, respectively.

T = 2.0 ps T = 0.2 ps
(a) traditional 2000 (b) (c)
etalon 1990
k1 k3
1980

1970
T t
1960
(d) time-reversed 2000 (e) (f)
etalon 1990
k 1 k3
1980
ωpump (cm-1)

1970
T t
1960
(g) Gaussian 2000 (h) (i)
k1 k3 1990

1980

T t 1970

1960
2000
(j) pulse pair (k) (l)
k1 k1 k3 1990

1980

τ T t 1970

1960
1950 1960 1970 1980 1990 2000 1960 1970 1980 1990 2000

ωprobe (cm-1)
 85

4.4 Time-domain method

Hinted from Fourier-transform spectroscopy, a narrow-band pump can be replaced with a pair of

transform-limited pulses whose time delay is scanned to generate ωpump via Fourier-

transformation. The method presented here more closely resembles the pulsed four wave mixing

method (8, 11, 33) except that the experiment is performed in a pump-probe beam geometry. In

this method, the pulse shaper is used to create two transform limited pulses. Rather than scan the

frequencies like was done above, the 2D IR spectrum is instead generated by collecting the

signal as a function of the time delay between the two pump pulses, τ, and Fourier transforming

it to give ωpump axis.

A mask function for a pulse pair is sum of “1” for one pulse at t = 0 and exp(iωτ) for

another pulse at t = τ (any function gets shifted in time by τ when its counterpart in frequency is

multiplied with exp(iωτ), then inversely Fourier-transformed), i.e.

1 iωτ
M (ω ) = (e + 1) (4.5)
2

Eq. 4.5 can be rearranged as

M (ω ) = cos(ωτ / 2)eiωτ / 2 . (4.6)

Thus, combining an amplitude mask of cos(ωτ/2) and a phase mask of ωτ/2 generate a pulse pair

composed of a fixed pulse and a scanning pulse with a time delay of τ. The mask in Eq. 4.5 sets

the phases of the two pump pulses inside their respective envelopes zero (φ1 = φ2 = 0), mimicking

the way that pulse delays are typically generated using translational stages.

With such a time-domain method, 2D IR spectra of W(CO) 6 in hexane were collected

and shown in the fourth row of Fig. 4.3 for τ = 0 to 10,000 fs in 14 fs steps. Like the frequency-
 86

domain methods in the Sec. 4.3, the spectra are automatically phased and have absorptive

lineshapes, but now the narrowest W(CO)6 pump linewidth is measured and there is no

discernable distortion in the peaks even at T = 200 fs (Fig. 4.3(l)). Moreover, the peak intensity

was ~75 times larger than the intensities measured with the narrow-band shapes when T = 2000

fs. This is because a pulse pair has more intensity as well as shorter duration than a narrow-band

pulse. Due to this, the 2D IR spectrum in Fig. 5d showed a new peak at 1955 cm-1 from the

second overtone transition (v=2-3).

Note that the time-domain method generated pure absorptive spectra with neither an

additional measurement nor a numerical calibration. In boxcar geometries, a rephasing and a

non-rephasing 2D IR spectrum are measured at the phase matching directions of − k 1 + k 2 + k 3

and + k1 − k 2 + k 3 , respectively, and show line shapes with wings tilted along the diagonal and

the anti-diagonal axes, respectively. When adding equally-weighted and well-phased rephasing

and non-rephasing spectra, the wings cancel out to yield a purely absorptive line shape that gives

the optimal spectral resolution (26). Therefore, in traditional four wave mixing experiments,

absorptive spectra ask for two 2D IR scans for rephasing and nonrephasing signals as well as a

transient absorption experiment to set time zeros (27). Once the spectra have been collected,

they must be phased and added. In a pump-probe geometry, since k 1 = k 2 , both the rephasing

and the non-rephasing signal are emitted collinearly and detected simultaneously while properly

weighted and phased automatically. Since the time-zeros are precisely zero, the desired

absorptive spectrum is obtained without any post data processing.

Femtosecond pump pulses allow linewidths closest to the intrinsic resolution to be

measured. We also point out that the peak shapes are symmetrical about ωpump and that there are

no spurious ghost images, indicating that the shaper time-resolution is sufficiently accurate.
 87

Asymmetric shapes and ghost images are common problems that occur when 2D IR spectra

are collected using translational stages to increment time delays (34, 35).

We usually use the time-domain method for most of applications. The time-domain

method is indeed the best way of collecting 2D IR spectra providing the best possible features:

intrinsic lineshapes, maximal signal strength, maximal time and frequency resolution, and

absorptive line shapes. Most uniquely, pulse shaping allows many new tricks of cycling and

incrementing phase that further enhance the pulse-pair method. So far, the phases of the pulses

in pulse pairs were held constant ( φ1 = φ2 = 0) to mimic the way that pulse delays are typically

generated using translational stages. However, it is straightforward for a pulse shaper to

arbitrarily set the phases and using this feature would make the automated 2D IR method truly

unique from others.

 88

4.5 Phase incrementing and phase cycling

Compared to the three frequency-domain methods in Sec 4.3, the time-domain method in Sec.

4.4 has the optimal time and frequency resolution in a straightforward manner. Furthermore, the

shaper allows arbitrary control over the phase so that 2D IR spectroscopy can borrow tricks that

have been extensively demonstrated in NMR spectroscopy including phase incrementing and

phase cycling. The pulse shaping mask in Eq. 4.5 for a pulse pair can be extended with

additional phase terms;

1
M (ω ) = [exp(iωτ ) exp(iφ1 ) + exp(iφ2 )] (4.7)
2

where φ1 and φ2 are the phases of the pulse at t = τ and 0, respectively. Any functional form in

frequency can be given for φ1 and φ2 . In a pump-probe geometry, two phase matching

directions of –k1+ k2+ k3 and +k1− k2+ k3 are detected, so the phase of the signal, φsig , can be

written as

φ sig = ± (φ1 − φ2 ) + φ probe − φ probe = ± (φ1 − φ2 ) = ± ∆φ (4.8).

Thus, the 2D IR signal depends on the relative phase between the pulses in a pulse pair, ∆φ .

Then, in Eq. 4.1, a phase term exp(i∆φ ) is convoluted with the response function and the 2D IR

signal changes its phase or frequency accordingly. Using this fact, we routinely implement

phase incrementing or phase cycling to shift the signal to a lower frequency, remove the

unwanted transient absorption background, and subtract scatter from the signal. For these

reasons, the pulsed method is the one most often used in our research group. In this section, we

outline each of these capabilities and describe the pulse sequence most often used in our group.
 89

4.5.1. Shifting signal frequency

In NMR spectroscopy, the signal frequency is shifted by incrementing the phase of radio-

frequency pulses proportional to the time delay so that fewer data points need to be collected

(36). This scheme is called as time-proportional phase increments. It can be easily implemented

to optical spectroscopy with a pulse shaper capable of phase control. In phase incrementing, the

phase of a femtosecond pulse is linearly dependent on the time delay, φ = ωiτ where ωi is the

frequency incrementing the phase. When ∆φ = 0 , the 2D IR signal oscillates in τ with ω0 , the

vibrational frequency of the system. When ∆φ = ωiτ , the 2D IR signal is convoluted with an

additional phase term of exp(iωiτ ) . Multiplying exp(−iω0τ ) with exp(iωiτ ) results in

exp(−i (ω0 − ωi )τ ) , thus the signal appears shifted in frequency at ω0 − ωi .

Shown in Fig. 6(a) are three time scans of n-methyl amide (NMA) in D2O at ωprobe=1625

cm-1 using three different ωi to increment ∆φ. In this particular case, we used φ1 = ωiτ and

φ2 = 0 . The ω0 of the amide I mode of NMA in D2O is 1625 cm-1, so when ωi = 0, the signal

oscillates with a period of 20 fs. When ωi = 1625 cm-1, then ω0 − ωi = 0 and the signal no longer

oscillates with τ. When 0 < ωi < ω0 , the signal oscillates with a period larger than 20 fs. In Fig.

6(a), ωi = 1200, 1400 and 1625 cm-1, so the period of the time scans increased accordingly (78 fs,

148 fs and infinity). These time scans were Fourier-transformed and their center frequencies

were measured in Fig. 6b. The ωi and the resultant signal frequencies were linearly correlated

with a slope of −0.99 and a root-mean-square of 2.0 cm-1, indicating that our shaper is

sufficiently accurate in incrementing phase.

In practice of FT spectroscopies, a continuous signal in nature is sampled to a numeric

sequence, a function of discrete time. An analogue signal that has been digitized can be perfectly
 90

reconstructed if the sampling increment in time, ∆t, is less than the half of the period of the

sinusoidal signal, ts (i.e. ts <∆t/2) (36). When this “Nyquist sampling criterion” is not satisfied,

the frequencies are overlap; that is, frequencies above 1/2ts will be reconstructed as and appear as

frequencies below 1/2ts. The resulting distortion is called aliasing. Aliasing not only confuses

the frequency, but also distorts the phase and the amplitude of the signal. Especially, the

sampled amplitude reduces when ts >> ∆t/2 because the “under”-sampling misses the top and the

bottom of the sinusoid. However, the undersampling is often used when the vibrational

relaxation takes too much time to use ts < ∆t/2 that enables “regular” sampling for data collection.

When the relative phase increments used to shift the observed frequency, the same ts that has

been used for undersampling can sample the signal without breaking the Nyquist criterion and

reducing the measured amplitude. This is particularly useful when a good signal-to-noise

spectrum should be recorded in a faster speed. For instance, to follow kinetics of amyloid

formation completing in a couple of hours (will be discussed in Chapter 5), a sampling scheme

using ωi of 1225 cm-1 and ∆t of 22 fs collects a 2D IR spectrum of the amide I band of the fibril

at 1618 cm-1 that relaxes in ~3 ps while spending only 0.4 sec for a single spectrum and keeping

the signal strength untainted (37).

 91

Figure 4.4 Sampling the amide I mode of NMA in D2O by incrementing the relative phase

in proportion to τ. (a) Time scans at ωprobe = 1625 cm-1 by using ωi = 1200, 1400 and 1625 cm-1.

(b) The measured frequency after Fourier-transforming the scans in (a) plotted with ωi used for

sampling.

(a)
30
∆OD at ωprobe=1625 cm-1

25 ωi =1200 cm-1

20

15 ωi =1400 cm-1

10

5 ωi =1625 cm-1

0 200 400 600 800 1000 1200 1400

τ (fs)

(b)
400
measured frequency (cm-1)

300

200

100

0
1200 1300 1400 1500 1600
ωi (cm-1)
 92

4.5.2. Removing background

According to Eq. 4.1, the 3rd-order signal is generated by two interactions with the pump pulse

(E1 and E2) and one interaction with the probe (E3). However, a 3rd-order signal is also generated

when E1 or E2 interacts twice with the sample, which gives rise to two transient absorption

spectra, e.g.,

( )
S B (τ , T , ω probe ) = ∫ dt ∑ Rn (τ , T , t ) ⊗ Ei* ⊗ Ei ⊗ Eprobe + Eprobe e
−iωprobet
(4.9)
n

where i=1 or 2 and ⊗ s represent convolutions of the electric fields with the response function.

Fig. 4.5 (as well as Fig. 4.4) outlines the three signals that arise from the two transient absorption

spectra and the desired 3rd-order spectrum: an exponential decay from the moving pulse (E1)

alone; a constant offset from the stationary pulse (E2) alone; an oscillation from E1 and E2. The

oscillatory part is the desired signal, but the exponential decay and the constant offset are

backgrounds of no interest. These background signals are easy to discriminate against in the

frequency domain, because neither oscillates when τ is incremented. However, in some cases

they can be a problem, such as when the signal frequency is shifted closer to ωpump=0 (when

working in the phase incrementing explained in Sec. 4.5.1, for instance).

The two transient absorption signals can be removed by programming all three pulse

sequences (E1 only; E2 only; E1 and E2) so that the two background signals can be subtracted

from the total signal. However, this effectively cuts the repetition rate of the laser system by 2/3.

A more efficient way is to use phase cycling. The phase of desired signal depends on the phase

difference between E1 and E2, e.g. ∆φ = φ1 − φ2, whereas the background transient absorption are

insensitive to the phase difference. Thus, for each time delay, we collect probe spectra with ∆φ =

0 and ∆φ = π, which we subtract, e.g.

 93

 I (∆φ = 0 ) 
∆OD = − log  , (4.10)
 I ( ∆φ = π ) 

instead of measuring a signal by tuning on/off the pump as in Eq. 4.3. Thus, background

subtraction is performed without a loss of repetition rate.

The subtraction procedure is demonstrated in Fig. 4.5, where we show the data for each

of the pulse sequences separately. Shown in Fig. 4.5(a) are two time scans of NMA in D2O at

ωprobe=1625 cm-1 when ∆φ = 0 (solid) and ∆φ = π (dotted). The oscillation when

∆φ = π appeared out of phase to the oscillation when ∆φ = 0, while the backgrounds remained the

same. Therefore, when the two scans with ∆φ = 0 and π were subtracted, the backgrounds were

removed; only oscillatory part remained and doubled in intensity (thick). Fig. 4.5(b) displays the

resultant spectra after FT giving a peak with the opposite sign for ∆φ = 0 (solid) and π (dotted)

containing an oscillatory noise along the entire ωpump and a peak doubled in intensity and free

from the oscillatory noise for the subtracted signal (thick). The oscillatory noise comes from the

constant offset because a step function in time generates oscillations along the entire frequency

after FT. Numerically subtracting the background in time domain after the experiment may

remove the oscillatory noise in frequency, but subtracting the two out-of-phase signals is

preferable because it also doubles the signal strength.

 94

Figure 4.5 Removing backgrounds from transient absorption using a phase cycling scheme

for the amide I mode of NMA in D2O. (a) Time scans at ωprobe = 1625 cm-1 when ∆φ = 0 (solid)

and ∆φ = π (dotted) and the subtracted product of the two scans (thick). (b) Fourier transforms

of the time scans in (a).

(a) 15

10
∆OD (x103)

0
∆OD(∆φ=0)
-5 ∆OD(∆φ=π)
∆OD(∆φ=0) − ∆OD(∆φ=π)
-10
0 500 1,000 1,500
τ (fs)
200
(b)
∆OD(∆φ=0)
150
∆OD(∆φ=π)
2D IR intensity (a.u)

∆OD(∆φ=0) − ∆OD(∆φ=π)
100

50

-50

-100
1400 1450 1500 1550 1600 1650 1700 1750 1800

ωpump (cm-1)
 95

4.5.3. Removing scatter

Phase incrementing or cycling allows scatter to be shifted or subtracted from the signal. Laser

light from the pulses can be scattered off spatially inhomogeneous samples such as crystals,

aggregates or any sample with length scales comparable to the wavelength of light. This is an

especially grievous problem with amyloid peptides, which we study in our laboratory, because

the peptides form fibrils spanning lengtzh scales of 30 nm to several microns (37). Fortunately,

much of this scatter can be removed by phase incrementing or cycling. The desired 3rd-order

signal arises from each pulse interacting with the sample once, while the scatter occurs from each

laser pulse, for i=1 or 2, separately, e.g.

S C (τ , T , ω probe ) = ∫ dt (Ei ⊗ Eprobe + Eprobe )e

−iωprobet 2
(4.11)

Thus, the phase of a background from the scattered pump depends on φ1 or φ2, whereas the signal

phase depends on ∆φ . If φ1 = φ2 = ωiτ , then ∆φ = 0 , then the signal remains the same in

frequency. In the meanwhile, the scatter phase is incremented by ωiτ and the scatter frequency

appears shifted at ω0 − ωi , where ω0 is the center frequency of the incident mid-IR.

Fig. 4.6 presents 2D IR spectra of amyloid fibrils from human Islet amyloid polypeptide

(hIAPP) in D2O. hIAPP forms fibrils ~100 nm in diameter and up to several microns in length

(37). As a result, the sample scatters the mid-IR pump, which interferes with the desired

spectrum. Scatter from the stationary pulse in the pulse-pair pump can be subtracted or filtered

because it appears at the ωpump = 0 (Fig. 4.6(a)). However, numerical subtraction is almost

impossible for the scatter from the moving pulse in the pulse pair because the scatter appear at

ωpump = ω0 as a broad line elongated along the diagonal axis that overlaps with the desired

spectrum (Fig. 4.6(b)). Rather than post-correcting our data, we shift the scatter frequency to
 96

separate the scatter from the desired spectrum during the experiment. When ωi = 1800 cm-1

was used to increment both φ1 and φ2, the scatter from the stationary pulse showed up at ωpump =

1800 cm-1; the scatter from the moving pulse appeared at ωpump = ~220 cm-1, elongated along the

anti-diagonal (Fig. 4.6(c)). The scatter lineshape is flipped because ω0 − ωi = −220 cm-1 and a

cosine function appears as mirror images at both the positive and the negative ωpump after FT.

Then, the desired 2D spectrum at ωpump = 1580~1680 cm-1 was completely separated from the

scatters shifted to ωpump = ~220 and 1800 cm-1 (Fig. 4.6(d)).

Instead of shifting scatter frequency with phase increments, phase cycling can be used to

subtract scatter from the signal. We collect four probe spectrum: I(φ1=0, φ2=0), I (φ1=0, φ2=π), I

(φ1=π, φ2=π), and I (φ1=π, φ2=0). We then add them to give ∆OD for each time delay:

 I (φ = 0,φ2 = 0 )   I (φ1 = π , φ2 = π ) 
∆OD = − log  1  − log  . (4.12)
 I (φ1 = 0, φ2 = π )   I (φ1 = π ,φ2 = 0 ) 

The result is that the scatter subtracts and the signal adds. This sequence also subtracts off the

unwanted transient absorption spectra discussed in Sec. 4.5.2. As a result, this is the series of

pulse sequence that we most commonly use when collecting 2D IR spectra. Since each pulse

sequence in the series is collected from one laser shot to another, it only takes 4 ms at 1 kHz

repetition rate to measure all four probe spectra. On this timescale, the scatter is essentially

constant, even for aggregating amyloids, so that the scatter subtraction can be done even when

studying protein folding (37).

 97

Figure 4.6 2D IR spectra of hIAPP fiber in D2O when using (a and b) no phase increments

and (c and d) ωi = 1800 cm-1. The left panel shows the entire ωpump from 0 to 2034 cm-1 and the

right panel highlights ωpump = 1580~1690 cm-1.

2000
(a) 1680 (b)
1600
1660
ωpump (cm-1)

ωpump (cm-1)
1200
1640

800 1620

400 1600

2000
(c) 1680 (d)
1600
1660
ωpump (cm-1)

ωpump (cm-1)

1200
1640

800 1620

400 1600

0
1580 1600 1620 1640 1660 1680 1580 1600 1620 1640 1660 1680
ωprobe (cm-1) ωprobe (cm-1)
 98

4.6 Rapid data collection

One of the most noticeable advantages of the automated method presented here is the far more

prompt data collection than traditional methods for colleting 2D IR spectra. In hole-burning

experiments, the center frequencies are adjusted by translating one of the two high reflectors in

an etalon. Because displacements of ~1 nm are needed to scan the center frequency by 1 cm-1,

piezo crystal actuators are typically used. Piezo crystal actuators are slow and not reproducible.

Thus, at each point during scanning, the pump pulse frequency is set using feedback from a

spectrometer, which is time consuming. In a traditional four wave mixing setup, the delay times

are adjusted by either translating a retroreflector to change the pathlength of the pulses to the

sample or translating a wedged optic to change the amount of material the pulses pass through.

For each translation, the computer spends 50-100 ms to communicate with the automatic stage.

To collect a typical 2D IR data set, each data point might be averaged for 50 or 100 laser shots,

which means that for a 1 kHz laser system, data collection is only half of as efficient as it could

be. The efficiency is even worse for higher repetition rate laser systems, where a larger fraction

of time will be spent moving translational stages.

In contrast, a pulse shaper can create pulse sequences without moving any optic on a

shot-to-shot basis, enhancing the efficiency of data collection drastically. For our germanium

acousto-optic modulator, it takes 10 microseconds for the sound wave to travel the length of the

modulator. Thus, a new waveform can be loaded every 10 microseconds, corresponding to a

repetition rate of 100 kHz. Thus, at 1 kHz, the time delay is easily incremented at every laser

shot, making data collection twice as fast with a pulse shaper. Furthermore, high repetition rate

has a much higher impact with pulse shaping 2D IR, because data collection rate decreases

linearly with repetition rate, unlike traditional methods in which the deadtime cannot be reduced.
 99

We typically utilize 426 waveforms for a single 2D IR spectrum. These waveforms

are uploaded into the memory of the arbitrary waveform generator prior to the experiment and

cycled continuously during the experiment. At each trigger from the laser, the waveform

generator cycles through the loaded waveforms, one at a time. Thus, at 1 kHz, an entire 2D IR

data set is collected in only 0.4 sec. This process is continuously repeated until the signal to

noise reaches a reasonable number. Averaging 100 shots (like in a traditional set), thus takes 40

s. The faster data collection is also expected to improve the averaging. Laser sources vary

across a range of timescales, from shot-to-shot fluctuations to minutes to hours. In FT

spectroscopy, a rapid-scanning scheme is well-known to limit noise sources to the shot-to-shot

noise of the light source by averaging out slow random fluctuations, which can contribute

significantly to the noise when scanned slowly (ref). Besides, most importantly, such a rapid

data collection allows monitoring dynamic events happening in seconds and minutes with 2D IR

spectroscopy on-the-fly without initiating the event for every frequency or time delay.

In practice, the memory of the arbitrary waveform generator limits the number of time

delays to collect a 2D IR spectrum. The number of time delays cannot exceed the half (or the

quarter when four laser shots are used for one ∆OD) of the number of loaded waveforms for

continuous cycling. When using 426 waveforms, the number time delays are 213 for regular

pump-probe signals (Eqs. 4.3 and 4.10) and 106 for the four-shot scheme in Eq. 4.12. For

tracking amyloid formation that scatters light extensively, we use four laser shots for each data.

Regarding the relaxation of amyloid fibrils (~2.5 ps), the increment of time delays should be

longer than 22 fs, which is larger than 20 fs, the period of a 6-micron mid-IR light. Rather than

undersampling, the signal frequency can be shifted using the phase increments in Sec. 4.5.1 so

that a 28-fs increment can sample the signal without violating Nyquist sampling criterion.
 100

Notice that the method can be tailored for each application by simply changing the software.

This is the most unique difference of the pulse shaping 2D IR comparing to traditional ways

which necessitates an extensive optical reconfiguration for any effort to enhance the method.

Therefore, a pulse shaper allows us to collect an ideal 2D IR spectrum in an extremely rapid

speed in a straightforward manner.

4.7 2D visible spectroscopy

Our method of collecting 2D spectra is not limited to the infrared. Our method of pulse shaping

in a partly collinear beam geometry is also applicable for generating 2D visible (2D Vis) spectra.

Pulse shaping has been used previously to collect 2D Vis spectra by generating fully collinear

pulse sequences (38) and by both spatially and temporally shaping for added control over

wavevector matching (39). However, 2D Vis spectroscopy can be straightforwardly implemented

using the protocols described here with a standard liquid crystal modulator and a transient

absorption optical layout (25). Regardless of which method is used, one of the biggest

advantages of using pulse shaping to collect 2D Vis spectra is that it overcomes one of the most

difficult challenges of implementing 2D Vis spectroscopy, which is the production of phase

stable pulse sequences.

To demonstrate the method, a 64-pixel liquid crystal modulator controlling both phase

and amplitude was used to shape a ~50 fs pulse at 800 nm running at a 1-kHz repetition rate.

The pulse pair is followed by an ultrashort probe pulse that is spectrally resolved. The delay

between the collinear pulses is incremented using phase and amplitude shaping and a 2D Vis

spectrum is generated by a following Fourier transformation. Shown in Fig. 4.7 are 2D Vis

spectra of atomic rubidium vapour. Rb vapor has atomic transitions at 794.76 (5P1/2 ← 5S1/2)
 101

and 780.03 nm (5P3/2 ← 5S1/2) and has been used to test other 2D E methodologies since it

has very narrow linewidths. The real part of the 2D E spectrum is shown in Fig. 4.7 and exhibits

diagonal peaks and cross peaks as expected. The diagonal peaks appear at λpump= λprobe= 780 and

795 nm along with crosspeaks between the two transitions. Shown in Fig. 10b is an expanded

plot of the real part for the diagonal peak at λpump= λprobe = 780 nm. Well-shaped lineshapes are

observed, indicating that the shaper can very accurately step the delay time. The linewidths

differ along λpump and λprobe, which is to be expected under our experimental conditions where the

linewidth along λprobe is determined by the natural linewidth convoluted with the spectrometer

resolution (~0.65 nm), while the linewidth along λpump is given by the natural linewidth

convoluted by the spectral resolution of the Fourier transform, 1/τmax, where τ max is the largest τ

and 2 ps here. We also measured the phase stability of the pulse pair generated by the shaper and

got λ/67 over the course of 35 hours. This number compares to phase drift of λ/100 typical of

diffractive optics and λ/250 with HeNe interferometry measurements made over the course of ~5

hours (40-42).

In most 2D spectrometers, each of the four pulses traverse an independent delay line

whose length must not vary more than a fraction of the wavelength during the experiment.

Achieving adequate phase stability with visible laser pulses can be difficult in conventional four

wave mixing methods. One approach for stabilizing the pathlengths is to use diffractive optics

and phase-compensating mirror arrangements (40). Another is to actively adjust the optical

pathlengths using piezocrystals and two sets of HeNe interferometers for feedback (42). If the

drift is slow enough, then it can be measured and the data corrected (43). All of these

approaches result in high quality multidimensional electronic spectra, but the extensive

infrastructure and effort required to implement these methods has discouraged all but a few
 102

dedicated groups from using this extremely powerful research tool. On the other hand, it is

straightforward to generate phase-locked pulse pairs with a pulse shaper capable of modulating

the spectrum of femtosecond pulses, like we have described above. Warren and coworkers

collected 2D Vis spectra by using an entirely collinear train of 4 pulses in analogy to the way that

2D NMR spectra are generated using phase cycling (38). However, the fully collinear geometry

necessitates the signal be measured by some observable other than absorption (e.g. fluorescence),

since the intense excitation pulses will saturate the detector in a linear absorption arrangement.

In contrast, Nelson and coworkers used a two-dimensional pulse shaper to collect 2D Vis spectra

in a non-collinear boxcar geometry where all 4 pulses in the pulse train impinge on the sample

from different directions (39). The approach by Nelson and coworkers takes advantage of phase

matching that provides background free signal and eliminates the need for phase cycling.

However, the two dimensional pulse shaper has very low throughput efficiency owing to the fact

that the input laser beam is spatially filtered to obtain the non-collinear geometry and few pixels,

which limits the complexity of the shapes that can be generated.

Our method that significantly eases the hurdles associated with collecting 2D electronic

spectra. 2D Vis spectra can be measured using commonly available pulse shapers and a partly

collinear pump-probe beam geometry. With a pulse shaper it is a trivial matter to make phase

stable pulse trains, thus eliminating difficulties associated with working at short (visible)

wavelengths. Furthermore, the beam geometry used in our experiments eliminates the need for

complicated phasing procedures. Our method eliminates many of the technical hurdles to

implementing 2D Vis spectroscopy, making it possible for many more research groups to exploit

this powerful spectroscopy in their research.

 103

Figure 4.7 2D electronic spectra of atomic Ru vapour: (a) real part over λpump=

λprobe=775~800 nm (b) expanded plots of real part for the diagonal peaks at λpump= λprobe=780 nm.

(a) 800 (b) 783

795 782

781

λpump (nm)
λpump (nm)

790
780
785
779

780
778

775 777
775 780 785 790 795 800 779 780 781
λprobe (nm) λprobe (nm)
 104

4.8 Conclusion

Femtosecond pulse shaping greatly enhances 2D IR spectroscopy with its ease of use, versatility

and speed. Programmable control of pulse shapes makes designing and implementing new pulse

sequences just a matter of computer programming. On a daily basis, this method is much easier

to maintain than a four-wave mixing setup, since the pulse shaper and the pump/probe geometry

requires minimal alignment. In many ways, the ease of alignment and accuracy of the collected

data is the greatest attribute of this method. In our experience, much more time can be spent on

the application of the technique to scientific questions rather than on technical maintenance of

the spectrometer and phasing of the data.

Rapid-scan 2D IR spectroscopy has been made possible by the development of a pulse

shaper that operates in the mid-IR. Further advances will be made possible by straightforward

improvements in technology. Polarization control has many uses in 2D IR and Vis

spectroscopies. In conventional pulsed photon echo experiments, controlling polarizations of all

four pulses selectively allows a combination of transitions at a specific order of angles (33).

Recently, we demonstrated a polarization scheme that assigns different polarization for the two

pump pulses collinearly overlapped in a Michaleson interferometer for improving the 2D IR

signal strength and eliminating unwanted background signals (44). It is now straightforward to

construct polarization pulse shapers in the visible/near-IR (45, 46), and it should be possible to

extend these approaches into the mid-IR. Coherent control methodologies can now also be

incorporated into multidimensional spectroscopies with simple programming. We showed that

mid-IR pulse shaping can be used to selectively enhance vibrational excitation of desired

quantum levels (47), which, if incorporated into a 2D IR pulse sequence, could be used to

enhance or eliminate spectral features (48). It should also now be straightforward to implement
 105

5th and higher-order signals to measure 3D spectra (49), for example, since addition of laser

pulses no longer requires complicated optical alignments but just additional programming. The

speed of rapid-scanning 2D IR spectroscopy can be further accelerated with high repetition rate

lasers. With current laser technology, it should be straightforward to increase data acquisition by

a factor of 30 or more, resulting in higher signal-to-noise spectra and faster kinetics. It is clear

that the utility of 2D spectroscopies for studying structures and dynamics will only improve in

the coming years.

4.9 Acknowledgment

We appreciate Prof. Niels Damruer and his group at University of Colorado at Boulders for the

collaboration that generated the 2D visible spectrum in Sec. 4.7.

 106

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 110

Chapter 5
Defining the Pathway of Amyloid Formation of human Islet
Amyloid Polypeptide with Residue-Specific Resolution

5.1 Introduction

More than twenty different diseases are associated with proteins that form insoluble amyloid

fibers (1-3). In large quantities, organ function is disrupted by the formation of amyloid deposits,

but for several amyloid diseases there is evidence that the toxic entities are actually pre-fibril

intermediates (4-6). However, details about these critical intermediates have been elusive,

mostly because it is extraordinarily difficult to obtain structural and kinetic information for

amyloid aggregation. The difficulty arises because high-resolution techniques do not have the

time-resolution required to track the structural changes nor can they be easily applied to

aggregating systems. Optical techniques that do have sufficient time-resolution, such as circular

dichroism spectroscopy, only provide a low resolution view of structure. Mechanistic

information is vital to understand the mechanism of protein mis-folding as well as to design

inhibitors that subvert the pathway of amyloid formation. Inhibitors which intervene early in the

process are particularly desired because they have the potential to prevent both fibril formation

and toxic prefibril intermediates (4-6). What is needed is a technique with sufficient time-

resolution to observe intermediates, provides residue-level structural information, and, ideally,

can be used to test molecular dynamics simulations.

A technique that satisfies these criteria is two dimensional infrared (2D IR) spectroscopy

when used with site-specific isotope labeling (7). In chapter 4, we demonstrated a new
 111

technological approach for collecting 2D IR spectra that is particularly well-suited for

studying amyloid formation (8). Our method uses a mid-IR pulse shaper to automate data

collection so that spectra can be collected quickly enough that fibril kinetics can be monitored

on-the-fly. In this chapter, we combine this automated version of 2D IR spectroscopy with site-
13 18
specific C O labeled peptides. Isotope labeling permits the kinetics of individual residues to

be measured, so that fibril formation can be followed on a residue-by-residue basis. The

resulting kinetics and secondary structure information provides a detailed pathway for the

formation of the fibril backbone.

In this chapter, we apply isotope-labeling and automated 2D IR spectroscopy to define

the pathway of amyloid formation in the 37-residue human islet amyloid polypeptide (hIAPP), a

peptide associated with type II diabetes. The deposition of amyloid fibrils in the islets of

Langerhans of the pancreas is a common pathological feature of type 2 diabetes and hIAPP is the

major protein component of these amyloid deposits (9-14). The polypeptide hormone is stored

with insulin in the secretory granules of the pancreatic β-islet cell and is secreted to the

extracellular space in response to the same factors that cause the release of insulin. Synthetic

amyloid aggregates are toxic to insulin-producing β-cells, arguing that hIAPP fibril formation

could play a role in the loss of β-cell mass in type 2 diabetes. The extent of amyloid deposition

appears to correlate with the severity of the disease, further highlighting a relationship between

amyloid deposition in the pancreas and the progression of the latter stages of type 2 diabetes (11-

14).
 112

5.2 Experimental

Peptide synthesis and purification. Peptides were synthesized on a 0.25 mmol scale using an

applied Biosystems 433A peptide synthesizer, using 9-fluornylmethoxycarbonyl (Fmoc)

chemistry. Pseudoprolines were incorporated to facility the synthesis as previously described

(15). 5-(4’-fmoc-aminomethyl-3’,5-dimethoxyphenol) valeric acid (PAL-PEG) resin was used to

form an amidated C-terminal. Standard Fmoc reaction cycles were used. The first residue

attached to the resin, β-branched residues, residues directly following β-branched residues and

pseudo-prolines were double coupled. Crude peptides were oxidized by dimethyl sulfoxide

(DMSO) for 24 hours at room temperature (16). The peptides were purified by reverse-phase

HPLC using a Vydac C18 preparative column. Analytical HPLC were used to check the purity of

the peptides before each experiment. The identity of the pure peptides was conformed by mass

spectrometry using a Bruker MALDI-TOF MS. 13C18O labeled Fmoc protected amino acids were

prepared as described (17). The level of 18O enrichment was at least 91% or greater and the 13C

enrichment was 98%. The high level of enrichment is critical to maximize signal to noise and to

avoid complication with 13C16O signals. The peptide was lyophilized in D2O and stored as a dry

powder.

Sample Preparation. The peptide was dissolved in deuterated hexafluoro-isopropanol (d-HFIP)

at a concentration of ~0.8 mM. An aliquot of the d-HFIP stock solution was evaporated under a

stream of N2 gas to generate a film. Amyloid formation was initiated by re-dissolving the film in

20 mM deuterated potassium phosphate buffer at pH 7. The final peptide concentration was 1

mM.
 113

Transmission Electron Microscopy (TEM). TEM images were taken of the 3 µL aliquot

of fibril solutions generated from three peptides that are unlabeled, Ala13, and Val32 labeled.

Fibril samples were prepared in the same way for the 2D IR spectra after incubating at least 3

days to complete aggregation. The aliquot was loaded onto a polyvinyl butyral coated copper

grid and stained with methylamine tungstate (Nanoprobes, Inc.). Excess stain was blotted off and

the sample was allowed to air dry. Images were acquired using a Philips CM 120 microscope

with an accelerating voltage of 80 kV.

Fluorescence and Fourier-transform infrared spectroscopy. Fluorescence data was taken

during aggregation of the peptide labeled at Ala13 using a Hitachi F-4500 fluorescence

spectrometer. The fluorescence shift due to binding of Thioflavin T to β-sheets was measured

with excitation wavelength at 450 nm and emission at 482 nm. Concurrent infrared absorption

data was taken using Bruker Optics Tensor 27 Fourier-transform infrared (FTIR) spectrometer.

The fluorescence data and growth of the β-sheet peak at 1618 cm-1 in the FTIR spectra are

compared in Fig. 5.2. Both the fluorescence and the FTIR data were measured with an average

time window of 100 seconds. The excellent agreement in the two different spectroscopic

measurements indicates that the unlabeled β-sheet feature at 1618 cm-1 is still an accurate

measure of β-sheet formation for isotope labeled peptides.

Automated 2D IR spectroscopy. The aggregation process, which took place between two CaF2

windows separated by a 100 µm Teflon spacer, was observed by the rapid scanning of four

phase-cycled pulse sequences. We create these 2D-IR pulse sequences using a Ge acousto-optic

modulator (AOM) based pulse shaper, as has been reported previously (18, 19). Briefly,
 114

femtosecond mid-IR pulses were initially generated by difference frequency mixing (DFM)

the two femtosecond near-IR outputs of a BBO-based optical parametric amplifier (OPA). A

ruled grating (150 g/mm) was then used to disperse the mid-IR pulses into the frequency domain.

The Ge AOM was used to modulate the phase and intensity of the frequency profile so as to

create the desired, phase cycled pulse pair. Rather than implementing a pump-probe experiment

in the most traditional sense, so that ∆OD = −ln(transmissionpump on/transmissionpump off), to

improve signal to noise and to get rid of the scatter from pump pulses, we took the difference in

transmission generated from four pump pulse trains with absolute phase shifted by π so that

∆OD = −ln(transmissionpump on, φ1 =0, φ2 =0/transmissionpump on, φ1 =π, φ2 =0) − ln(transmissionpump on,

φ1 =π, φ2 =π/transmissionpump on, φ1 =0, φ2 =π). The waveform generator for the pulse shaper can

continuously cycle 426 pulse shapes at maximum, thus we can scan 106 time delays when using

four phase cycles. To scan over >2 ps of the first coherence time with the 106 time delays, the

relative phase of the pump pulse pair was cycled with a rotating frame frequency of 1225 cm-1,

which enables regular sampling with a 22 fs step. It took 0.43 seconds to measure a single 2D IR

spectrum at a 1 kHz repetition rate. A running average was then performed in the same way for

all experiments until adequate signal to noise was achieved. The 2D IR spectra shown in Figs.

5.4, 5.6, 5.7, and 5.8 are averaged over a 9 minute window while the kinetic traces are averaged

over a 18 minute window. There is ~2 minute dead-time between initiation of the aggregation

and the collection of the first 2D IR spectrum.

Isotope dilution. Samples of mature fibers, whose isotope-label diluted to 25%, were prepared

by mixing the d-HFIP stock solutions of the labeled and the unlabeled peptides, evaporating d-

HFIP, re-dissolving in 20 mM phosphate buffer at pH 7, placing the aqueous solution between

 115

two CaF2 windows, and incubating in room temperature for 2 hrs at least. The concentration

of the labeled peptides was 1 mM for both the 25% and the 100% label samples. Three sets of

samples, along with a 100% unlabeled peptide, were prepared in the same way for Ala13, Ala25

and Leu27. Each set composed of a 100% and a 25% labeled peptide. >5,000 of 2D IR spectra

for the seven samples were collected, averaged and sliced through the diagonal to give Fig. 5.10.

The diagonal intensities of each sample were normalized with the intensity of the unlabeled β-

sheet feature at 1618 cm-1, then subtracted from the diagonal slice from the 100% unlabeled

sample.

5.3 Kinetics & morphologies during amylodosis: Fluorescence, FTIR

and TEM images

To delineate the pathway of amyloid formation in a pathologically relevant condition, we used

the wild-type hIAPP in the full length (37 residues). Many of previous studies rely on small

peptide fragments, which do not necessarily translate into the context of the full protein. For

example, studies using fragments of hIAPP proposed that residues 20 through 29 form the core

of the cross b-structure of the fiber (20), but recent solid-state NMR (ssNMR) studies of full-

length hIAPP reveal that this is not the case (21). Furthermore, the kinetics of amyloid formation

by short fragments of hIAPP differs dramatically from that observed for the full length hormone.

The same difficulties arise from studies of fragments of the Alzheimer’s Aβ1-40 and Aβ1-42

peptide. For these reasons, we have chosen the full 37-residue peptide.

The polypeptide hormone is stored with insulin in the secretory granules of the pancreatic

β-islet cell in a concentration of 0.5~2 mM. Such a high concentration is also necessary for a
 116

single carbonyl (i.e. a single residue isotope-labeled) to produce reasonable signal strength at

the amide I mode in FTIR and 2D IR spectra. To begin with unstructured state in such a high

concentration, amyloid formation was initiated by the addition of buffer to dried films of

unstructured hIAPP. According to transmission electron microscopy (TEM) images (Figs. 5.1A

to C), the fibrils produced using this protocol have the same structure as those formed using

other methods (22). Figs. 5.1A to C show a common morphology of long fibrils with a mean

diameter 10 nm for all the three fibril samples prepared from three differently labeled peptides

(unlabeled, labeled at Ala13 and labeled at Leu27). Moreover, kinetic curves generated from

Thioflavin T (ThT) fluorescence and from IR absorbance of the β-sheet are sigmoidal, as is

typical of amyloid kinetics (Figs. 5.2B). The fluorescence shifts due to binding of ThT to β-

sheets, thus increasing trend of the emission measured at 482 nm reflects the growing

populations in fibril. The amide I band of the FTIR spectrum right after initiation gave a broad

peak centered at 1644 cm-1, indicating that the dominant structure was random coil at the

beginning. As aggregation progressed, the random coil feature at 1644 cm-1 decreased and the

antisymmetric stretch of β-sheets located at 1618 cm-1 increased (Fig. 5.2A). Thus, FTIR spectra

indicate the structural progression of the peptide from the soluble state in random coil to the

insoluble fibril state rich in β-sheet during amyloid formation.

Concurrent to the fluorescence experiment, TEM images were taken from four aliquots

removed from the sample, diluted and flash-frozen at four times during aggregation to monitor

morphological changes (Figs. 5.3). The TEM image taken immediately after initiation of the

reaction showed predominantly small 13 to 16 nm spherical oligomers with trace numbers of

fibers (Fig. 5.3B). At t = 20 min, the kinetics curve began to leap and we observed large

spherical oligomers in diameters of 20~35 nm as well as short fat fibers (~20 nm wide) (Fig.
 117

5.3C). After the kinetics curve slightly passed the time-to-half-maximum (t50), the TEM

snapshot showed aggregates of short fat fibers and networks of long thin fibers (final fiber

structure) concentrated mostly on the left and right of Fig. 5.3D, respectively, free from spherical

oligomers. After ~4 hrs, the amyloid formation was completed and only the final fibers in a ~10

nm diameter were found in Fig. 5.3E similar to the fiber structures shown in Fig. 5.1. From the

TEM snapshots, we suggest a morphological progression in amylodosis starting from spherical

oligomers whose their diameter increase to reach a critical point that interconnect the granules

into short fat fibrils, which transform into the final structure of long and thin fibrils. Therefore,

not only the secondary structure evolves, but also the macroscopic morphologies progress during

amyloid formation.
 118

Figure 5.1 Morphologies of hIAPP aggregates from three different batches of

synthesized peptides with no labeling(A) and with isotope-labeling at Ala13(B) and Leu27(C).

The three TEM images share common motif of fibrils in a diameter of ~10 nm and lengths that

are a fraction of a micrometer.

A B C

100 nm
 119

Figure 5.2 Kinetic studies of amyloid formation of hIAPP isotope-labeled at Leu27 at

500 µM in 20 mM phosphate buffer (pH 7.0). (A) FTIR spectra of the amide I band at t = 4 min

(solid), 60 min (dotted), 120 min (dashed) and 300 min (thick solid). (B) Kinetics curves from

intensities of the anti-symmetric β-sheet feature at 1618 cm-1 and fluorescence intensities of

Thioflavin T.

0.03
A
absorption (a.u.)

0.02

4 min
60 min
0.01
120 min
300 min

0.00
1580 1600 1620 1640 1660 1680 1700
frequency (cm-1)

1.0
B
Normalized Intensity

0.8

0.6

0.4

0.2
ThT fluororescence
IR absorbance of the β-sheet
0.0

0 20 40 60 80 100 120 140 160 180 200

t (min)
 120

Figure 5.3 Concurrent ThT fluorescence and TEM snapshots during amyloid formation

of hIAPP isotope-labeled at Leu 27. (A) Kinetics curve generated from fluorescence. Circles

indicate the time points of the four aliquots removed from the sample for TEM snapshots. (B to

E) TEM snapshots taken from the four samples removed at t = 3, 20, 57 mins and ~4 hrs after

initiation of amyloid formation. All four images are scaled in the same way.

A
fluorescence intensity

1.0
E
0.8
D
normalized

0.6
0.4
0.2 TEM
B C
0.0
ThT Fluorescence
0 20 40 60 80 100 120 140 160
time after mixing (min)

B C

100 nm

D E
 121

5.4 Secondary structures during amylodosis: 2D IR spectra of

unlabeled hIAPP

The FTIR spectra collected from hIAPP transforming into amyloid fiber contain all secondary

structural information during the process. However, only the β-sheet feature could produce a

kinetics curve without deconvolution, which is prone to error. The random coil feature at 1644

cm-1 is hard to separate from the final IR spectrum of the fiber (Fig. 5.2A), which contains

unignorable amount of absorbance around 1644 cm-1. This is a common problem of one-

dimensional spectroscopy including circular dichroism spectroscopy to study amyloid formation

kinetics since the spectral features of interest all absorb in the same wavelength range. If the

overlapped spectral features are spread over two dimensions, they could be separately monitored

without erroneous deconvolution. 2D IR spectroscopy has the potential to improve our structural

knowledge of amyloid formation due to its increased structural resolution and its femtosecond

time resolution.

Despite the advantages of 2D IR spectroscopy, there are two hurdles to applying 2D IR

spectroscopy to amyloid formation. First, amyloid fibers as well as intermediates scatter light,

which creates a large background signal. Second, the aggregation kinetics of amyloids is poor in

reproducibility, so that it is not possible to initiate the reaction over hundreds times to collect 2D

IR spectra at various stages of amylodosis. We overcome these two obstacles by using the

automated method for 2D IR spectroscopy using a mid-IR pulse shaper. Our method removes

scatter by incrementing or cycling phase described in Sec. 4.5.3 and collects a single 2D IR

spectrum in <1 sec as explained in Sec. 4.6. Thus, we can directly track the amyloid formation

of hIAPP on-the-fly.
 122

Shown in Figs. 5.4A to C are three representative 2D IR spectra generated from many

thousands of spectra collected during fiber formation. Each peak in a FTIR spectrum produces a

pair of peaks in the opposite sign in a 2D IR spectrum because both fundamental and overtone

bands are probed. At the beginning of the amyloid formation, the most noticeable feature is an

out-of-phase doublets with the negative peak at ωpump = ωprobe = 1644 cm-1. The peak position

and shape (broad, diagonally elongated) indicate that the peptides are predominantly random coil

with a wide distribution of structures. As aggregation proceeds, the random coil doublet gives

way to a doublet at ωpump = ωprobe = 1618 cm-1 with a narrow linewidth of 15 cm-1 that is created

by the antisymmetric stretch of the β-sheet amyloids. At ωpump = ωprobe = 1673 cm-1, the β-sheet

symmetric stretch gives a weak doublet. IR absorbance between 1618 and 1673 cm-1 come from

residues which do not form regular β-sheet in the matured fiber.

Populations of random coil and β-sheet were monitored from the two features in the 2D

IR spectra without deconvolution. Kinetics curves in Fig. 5.4D were generated from intensities

of two features in 2D IR spectra: fundamental sequence peak of β-sheet at ωpump = ωprobe = 1618

cm-1; overtone sequence peak of random coil at ωpump = 1644 cm-1 and ωprobe = 1624 cm-1. The

two features are marked with a solid and a dashed arrow on Fig. 5.4B for the β-sheet and random

coil features, respectively, chosen for the kinetics curves. At the position for the random coil, β-

sheets contribute negligibly, as can be seen at t = 120 min, when the fibers mature and the

overtone intensity peak has gone away. The kinetics curves of the two features (Fig. 5.4C) are

sigmoidal, typical of amyloid kinetics, as in kinetics curves from ThT fluorescence and FT IR

spectra (Fig. 5.2B). However, the kinetics curves for β-sheet and random coil populations differ

in the t50, the rate of rise and the length of lag phase. The difference between the two kinetics is

more noticeable when normalized and smoothed as in Fig. 5.4E. In our previous study of hIAPP
 123

amylodosis in pure D2O at pH ~6, the kinetics of random coil and β-sheet were also

significantly different, although both of them had no lag phase. The random coil kinetics fit to a

single exponential whereas the β-sheet kinetics fit to a biexponential (8).

The different kinetics of random coil and β-sheet indicate that the amyloid formation

cannot be explained with a simple two-state model. As shown in Fig. 5.4E, the lag phase of

random coil is almost half of the β-sheet lag phase, indicating that some intermediates exist

during the intermission. The intermediates, neither in random coil nor in the final β-sheets, may

consume random coils, then remain for ~20 minutes, and then transform into mature fibrils. In

Sec. 5.3, TEM images revealed intermediate morphologies including spherical oligomers and

short fat fibrils that disappear once the fibril fully mature. These macroscopic intermediates may

have secondary structures emitting unnoticeable amount of signal or overlapping with the

dominant features in the 2D IR spectra, but contributing to the lag phase and the rise time of the

kinetics curves of random coil and β-sheet. In Aβ40 amylodosis, a ssNMR study suggested that

random coils aggregate into small spherical oligomers, then transform into large spherical

oligomers (15-35nm) composed of β-sheets prior to the final fibril state (23). To identify

intermediate structures of hIAPP amylodosis, we need to further resolve the amide I band of the
13
peptide. Isotope-labeling with C=18O separates a single residue or a number of residues in

frequency by ~50 cm-1 away from the rest of peptide backbone composed of 12
C=16O. In the

next section, on-the-fly 2D IR experiments of isotope-labeled peptides will be introduced.

 124

Figure 5.4 Representative 2D IR spectra and kinetics curves of hIAPP during aggregation.

(A to C) The 2D IR spectrum at (A) t = 5 min, (B) 40 min, (C) 120 min. (D) Kinetics of the

diagonal peaks of the β-sheet at ωpump=ωprobe=1617 cm-1 and the random coil at ωpump=1645 cm-1

and ωprobe=1624 cm-1. 500 spectra were averaged. (E) Normalized and smoothed kinetics curves

from the kinetics curves of the β-sheet and random coil features in D. 2,000 spectra were

averaged to give each point.

1680 A B C
1660
ω1 (cm-1)

1640

1620

1600
1580 1620 1660 1700 1620 1660 1700 1620 1660 1700
ω3 (cm-1) ω3 (cm-1) ω3 (cm-1)

50
D 100 E
40
normalized intensity
peak intensity (a.u.)

80

30 60
β-sheet β-sheet
20 40
random coil random coil
20
10
0
0
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
time (min) time (min)
 125

5.5 Residue-specific assemblies during amylodosis: 2D IR spectra

of isotope-labeled hIAPP

5.5.1 Six residues labeled

To monitor the structures and kinetics of individual residues in hIAPP, we use 13C18O labeling of

the backbone carbonyl groups to resolve single amide I stretches (24). Isotope labeling and IR

spectroscopy has been used previously to study amyloids, which have provided insight into

general aggregation mechanisms (25, 26).

13 18
Six separate samples of hIAPP were prepared, each containing a single C O labeled

residue (21). The positions of the six labeled residues are shown in Fig. 5.5 superimposed upon

the recent model of the hIAPP protofibril deduced from solid state NMR (21). hIAPP forms a

cross β-strand structure that is composed of two twisted columns of peptides. According to the

ssNMR studies, each column is created from a stack of peptides that are folded into a structure

containing two β-strands with an ordered loop between residues 18 and 27. Hydrogen bonding

occurs along the stacks so that there are four β-strands running in parallel along the lengths of

the fibrils. The labeled residues are spaced along the peptide (Fig. 5.5) and the positions were

chosen to provide probes throughout key regions of the fibril. Note that residues 1-7 do not form

part of the ordered core of the fibril, in part because there is a disulfide bond between Cys2 and

Cys7 that is not compatible with β-sheet structure. Since the peptides are stacked, each isotope

label forms two columns, illustrated in Fig. 5.5C for Ala25. The kinetics we report below

reflects the arrangement of the isotopes into their respective columns.

 126

Figure 5.5 Structure of hIAPP fibrils according to solid state NMR. (A) Sequence of

hIAPP with the six isotope-labeled residues highlighted: Ala8 (yellow), Ala13 (blue), Val17

(purple), Ala25 (red), Leu27 (cyan), and Val32 (green). (B) Cross-section through a fibril,

illustrating that the fibrils are composed of two columns of peptides to create four β-strands (that

run in and out of the page). The labeled residues are highlighted. (C) Side-view of the

protofibril illustrating how a peptide with a single labeled residue creates two columns of isotope

labels that run along the β-sheets.

A
1 6 11 16 21 26 31 36
KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

B C
 127

5.5.2 2D IR spectra and kinetics of hIAPP labeled at Ala 25

Shown in Fig. 5.6 are four 2D IR spectra collected at successively longer times during amyloid

formation for a sample of hIAPP labeled at Ala25. The four spectra are generated from many

thousands of spectra that were collected. The first 2D IR spectrum in Fig. 5.6 is at t = 5 min,

which is the first averaged 2D IR spectrum collected after aggregation is initiated. The most

prominent features in these spectra are between 1617 and 1670 cm-1 that are due to the unlabeled

residues (inside the dashed black squares) as shown in Sec. 5.4. The unlabeled peaks give

information on the overall assembly kinetics of the amyloid (8). At t = 5 min., the unlabeled

features consists of two out-of-phase peaks at ωpump = 1645 cm-1, typical of random coil peptide

structures with large structural distributions. The isotope labeled features appear near 1580 cm-1

(inside the solid black squares), which provides information specific to the folding kinetics of

Ala25. At t = 5 min., the isotope labeled absorption is very broad and weak, indicating that

Ala25 is conformationally disordered, which is consistent with the nature of the random coil

state.

To highlight the changes which occur in the 2D IR spectra during the folding processes,

the remaining 2D IR spectra in Fig. 5.6 are plotted as difference spectra, calculated by

subtracting the first 2D IR spectrum in Fig. 5.6A from the others. As time progresses, the

random coil doublet disappears while a doublet grows in at ωpump = 1618 cm-1, the signature of β-

sheet amyloid. Concurrent with the growth of the β-sheet, two isotope labeled features appear at

1574 and 1585 cm-1. The lower frequency peak eventually becomes more intense (Figs. 5.6D

and 5.7A). Cross-peaks also grow in between the isotope labels and the unlabeled β-sheet peak

at 1618 cm-1, indicating that Ala25 is strongly coupled to the β-sheets. Thus, the cross peaks

measure the kinetics of A25 folding into the β-sheet configuration of the amyloid fibril. Figure
 128

5.7B plots the intensity of the peaks for the unlabeled β-sheet, Ala25, and the cross peak as a

function of time. All three kinetic curves are sigmoidal, as is typical of amyloid kinetics, and all

three have a time-to-half-maximum (t50) that is virtually identical, indicating that when Ala25

becomes part of the ordered fibril, it assembles directly into a β-sheet structure.
 129

Figure 5.6 Representative 2D IR spectra of hIAPP labeled at Ala25 during aggregation.

(A) The first averaged 2D IR spectrum at t = 5 min. (B to D) Difference 2D IR spectra at (B) t =

31 min, (C) 66 min and (D) 187 min, calculated by subtracting the t = 5 min. spectrum. Insets

highlight the diagonal peaks from 13C18O Ala25. Contours and color scales are set in the same

way for B to D. Black boxes surround spectral features of the unlabeled portion of the peptide

while red boxes enclose the diagonal peaks of the isotope labeled Ala25. Blue and green arrows

highlight the two labeled features, while the red arrows points to the cross peak between the
13 18
C O Ala25 and the unlabeled β-sheet.

10 5 0 −5 −10

1680 A
ωpump (cm-1)

1640

1600

1560
1560 1600 1640 1680
ωprobe (cm-1)
4 3 2 1 0 −1 −2 −3 −4

1680
B C D
ωpump (cm-1)

1640

1600

1560
1560 1600 1640 1680 1560 1600 1640 1680 1560 1600 1640 1680
ωprobe (cm-1) ωprobe (cm-1) ωprobe (cm-1)
1600 1600 2 1600
1
1580 1580 0 1580
−1
1560 1560 −2 1560
1560 1580 1600 1560 1580 1600 1560 1580 1600
 130

Figure 5.7 Kinetics curves of hIAPP labeled at Ala25 generated from 2D IR spectra during

aggregation. (A) Kinetics of the diagonal peaks of the unlabeled β-sheet at 1617 cm-1 and the

two label features (black and blue arrows). (B) Kinetics of the cross peak are compared to the

diagonal peaks. The left y-axis is for the β-sheet feature at 1617 cm-1 and the right y-axis is for

other features.

200
16
A B
β-sheet Intensity

160

label Intensity
12
120
8
80
β-sheet β-sheet 4
40 label (1583 cm -1) label (1583 cm -1)
label (1576 cm -1) crosspeak
0 0
0 20 40 60 80 100 0 20 40 60 80 100
t (min) t (min)
 131

5.5.3 Reproducibility of the kinetics

The reproducibility of our experiments are particularly important issues with regards to amyloid

formation and this is one of the first detailed studies of amyloid kinetics using 2D IR

spectroscopy. It is well-known that amyloid kinetics are not perfectly reproducible from one

experiment to the next because they are extremely sensitive to conditions which are difficult to

control, such as how rapidly the solution is mixed (27). Procedures for comparing separate data

sets have been established using fluorescence and CD spectroscopy. Our procedure is similar to

these well-established methods, but accounts for issues specific to infrared spectroscopy. We

investigated the reproducibility of our 2D IR kinetics experiments by repeating multiple runs

with the same peptide labeled at Ala25 prepared in the same condition. Shown in Fig. 5.8A are

the kinetic traces of the unlabeled β-strand feature from four separate 2D IR experiments on the

same sample of hIAPP labeled at Ala25. The time-to-half-maximum (t50) times for these 4

experiments fall between 30 and 60 min. This large range of times prohibits the averaging of

successive 2D IR data sets, which is why our rapid-scan version of 2D IR spectroscopy is

necessary to accomplish these experiments. Fortunately, it has been established from

fluorescence studies that kinetic scans on wide ranging timescales can be compared by scaling

the time axes with their respective t50, so long as the nucleation mechanism is unchanged.(28)

Following this procedure, we first established the value of t50 for each scan by fitting the kinetic

traces associated with the spectral features due to the unlabeled 12C16O oscillators to a sigmoidal

function

 1   1 
f (t ) = [r1 − m1 exp(−t / τ )]  + (r1 + m1t )1 − 
 1 + exp(−(t − t 50 ) / σ )   1 + exp(−(t − t 50 ) / σ ) 

(5.1)
 132

where the exponential decaying function of r1− m1exp(−t/τ) defines the upper baseline and

the linear equation r1+ m1t defines the lower baseline. Flat baselines were used for the kinetics

curves with low signal-to-noise ratio. Fits were performed using a nonlinear algorithm in

MatLab. For fitting, we used 2D IR data averaged over a 1 minute window. (In Fig. 5.8, we

smoothed the data (dots) and the fitted curves (solid lines) with a 18 mins window for

visualization purpose after fitting.) We then used the t50 determined by fitting to scale the time

axes.

On this scaled axis, the sigmoidal curves are quite similar (Fig. 5.8B). The similarity of

the slopes indicate that all 4 experiments are monitoring the same aggregation mechanism, as is

expected from identical samples. Having determined the scaling parameters from the unlabeled

β-strand features, we then scale the time course of the spectral features associated with the

labeled site using the same parameters. For example, Fig. 5.8C shows the time course of the

Ala25 signal at 1585 cm-1 for the same 4 experiments after scaling with the t50 times used above,

along with the sigmoidal fits. The signal-to-noise of the label kinetics is lower than the

unlabeled β-strand features because the label intensities are about 13 times weaker.

To account for signal-to-noise, we calculate the error in the ratio of t50s between the label

and the β-strand features. To estimate the error, the vector containing standard errors of the

fitted parameters (σ) were calculated from the sum of (residuals)2 and Jacobian matrix (J) using

σ = diag (∑ (residual ) 2 ( J T J ) −1 ) (5.2)

where “residuals” are the difference between the original data and the fitted data and J is the

matrix of all the first-order partial derivatives of the fitting function with all the fitted parameters.

The scaled data is calculated by dividing by the t50 of the unlabeled β-sheet feature
 133

tl
tn = (5.3)
tb

where tn is the normalized t50, tl is the t50 of the labeled kinetics and tb is the t50 of the unlabeled

β-sheet kinetics. The standard error in the scaled t50 times (σn) accounts for error in both the

labeled and unlabeled β-sheet kinetics (t1 and tb) using:

2
 dt n   dt n 
2
 2
σb  
2
2 σl 
σ =  σ l  +  σ b  = tn   +   
2
n (5.4)
 dtl   dtb   tl   tb  

where σl is the error in tl and σb is the error in tb.

The unlabeled β-sheet kinetic traces usually have a slow rise after the fast transition,

which is commonly observed in amyloid studies using ThT fluorescence (28). The variation of

the slow rise between experiments is illustrated in Fig. 5.8D for the four Ala25 trials. Our fitting

function accounts for this rise with a simple exponential function and the fits predict that it has a

minor contribution to the t50 of the sigmoidal transitions because it occurs on a much longer

timescale than the fast sigmoidal kinetics. The difference in time scale is readily apparent in the

Ala25 data for which both isotope label peaks exhibit nearly identical fast kinetics but very

different slower kinetics. Even so, fits give both Ala25 peaks nearly identical t50 times of 49.24

and 49.51 mins for 1585 cm-1 and 1574 cm-1 peaks, respectively, for the experiment shown in

Figs. 5.6 and 5.7. Of course when the data is plotted in the figures the kinetic traces must be

normalized. We chose the relative time t/t50 =2 (e.g. Fig. 5.7, 5.8 and 5.12). The choice of

normalization time has no bearing on the reported t50 data which comes directly from the fits

themselves. The choice of normalization does slightly alter the slopes of the kinetic traces at t50,

but we know that the effect is small by comparing the sigmoids from the fits with the additional
 134

exponential function, shown in Fig. 5.8D for the 4 Ala25 trials. Thus, our fitting routine is

robust with regards to the t50 and the slopes visualized in the manuscript figures are accurate.

The resulting ratios of t50 and their standard error bars (σn) are plotted in Fig. 5.8E. All 4

measurements give a very similar ratio, with much smaller error bars associated with the higher

quality data sets, as expected. Taken together, these 4 measurements give a combined t50(label)/

t50(unlabeled) = 0.99 ± 0.13 with 90% confidence for Ala25.

 135

Figure 5.8 Summary of experiments using Ala25 labeled peptides to test reproducibility

and data analysis method. (A) Kinetics of unlabeled β-strand features at 1617 cm-1 for 4

independent experiments (dots), along with sigmoidal fits (solid line) using Eq. 5.1. Data and

fits are smoothed with a 18-min. window after performing fits on data averaged with a 1 minute

window. (B) Curves from (A) scaled by their respective t50 times. (C) Kinetics of Ala25

feature at 1585 cm-1, using the same t50 times along with sigmoidal fits. (D) Curves in (B)

shown in longer time scale. The intensities are normalized to the fitted value at the relative time

t/t50 =2. The experimental data in dots are shown along with its fits in solid line with the same

color for each experiment. (E) t50 times and standard errors extracted from fits of isotope

kinetics shown in (C). Colors are assigned in the same way for each of the 4 experiments.

A B C
normalized intensity (a.u.)

1.2
1.0
0.8
0.6
0.4 exp1 exp1 exp1
exp2 exp2 exp2
0.2
exp3 exp3 exp3
0.0 exp4 exp4 exp4
-0.2
0 20 40 60 80 100 0.2 0.6 1.0 1.4 1.8 0.2 0.6 1.0 1.4 1.8
t/t50 t (min) t/t50
1.2
normalized intensity (a.u.)

D E
t50 (label) / t50 (unlabeled)

1.0
1.1
0.8

0.6
1.0
0.4
exp1
0.2 exp2 0.9
0.0
exp3
exp4
-0.2 0.8
0 1 2 3 4 1 2 3 4 Avg
t/t50 repeats of experiments
 136

5.5.4 Kinetics of six labeled sites

The kinetics of the other five labeled sites were also measured. They have similar spectral

features to Ala25 (Figs. 5.9 and 5.10), but strikingly different kinetics; the kinetic transition

measured for some labeled sites is more rapid than the transition monitored by the unlabeled β-

sheet peak, while others fold much slower. To better compare the relative folding times, we

have fitted the kinetics curves with sigmoids as in the previous section (Data and fits shown in

Fig. 5.11. Function given by Eq. 5.1), then scaled the kinetic curves for the unlabeled β-sheet

peaks from all 7 samples (6 labeled and 1 unlabeled peptides), so that their t50 times are equal

(Fig. 5.12A). Scaling is necessary because amyloid kinetics are not perfectly reproducible from

one experiment to the next, which is why our rapid-scan version of 2D IR spectroscopy is

necessary to accomplish these experiments. However, the kinetics from different experiments

can be compared by dividing the time-axes by the respective t50 measured for the unlabeled

peaks (28) as we demonstrated for multiple experiments of the same peptide labeled at Ala 25 in

the previous section. We find that when the axes are scaled, the unlabeled β-sheet peaks for all

six labeled peptides have virtually the same folding curve and that this folding curve matches

that observed for an unlabeled peptide without any isotope labels (Fig. 5.12A). The slopes show

slightly more variation (mostly because of Ala8 and Ala13) than for multiple runs of the same

sample (Fig. 5.8B), which may be caused by small differences in HPLC purification of the

peptides, but is not large enough to indicate a significantly different aggregation mechanism (28,

29). Thus, we conclude that the kinetics from individual 2D IR measurements can be compared

by following the standard analysis methods of fluorescence experiments by using the unlabeled

β-sheet feature as a reference. The β-sheet feature provides an internal standard which allows

one to deconvolve the normal run-to-run variations from the more interesting differences
 137

associated with the kinetics of assembly. The presence of such a built in internal standard is

an added bonus of the 2D IR technique.

Having established the validity of our 2D IR approach, we now turn to comparing the

kinetics of all 6 labeled residues, which is the primary motivation for this study. The scaling

parameters in Fig. 5.12A are then used to compare the relative folding rates of the isotope labels,

which are shown in Fig. 5.12B. Simple visual inspection of this data reveals that these 6 residues

exhibit strikingly different kinetics; some residues transition slower than the unlabeled β-strand

feature, while others are faster. The data shown in Fig. 5.12B are solely presented to visualize the

differences in kinetics. Like Ala25, each labeled peptide was measured multiple times and the

t50 were calculated from the best 2 to 4 data sets (Fig. 5.12C). The averaged t50 times are plotted

in Fig. 5.12D. Fig. 5.12D exhibits a trend in which the first residues to transition are near the

middle of hIAPP sequence, followed by aggregation at the ends. Val17 is the first residue to

transition and has a t50 that is 10% sooner than the t50 of the unlabeled residues (in all

experiments performed, the Val17 isotope label rises before the unlabeled β-sheet features),

while Val32 is the last to transition with a t50 time that is 30% slower. On an absolute timescale,

this 40% difference in folding rates translates into a 20 min. difference between the folding of

Val17 and Val32 for a typical t50 folding time of 50 minutes.

From the scaled t50s of 6 residues, we estimated the order in which each residue

assembles into the final structure. The first residue to fold is Val17, on the N-terminal end of

the ordered loop, followed by Ala25 on the C-terminal end of the loop. The folding then

proceeds down the β-strands, although at unequal rates towards the two termini. hIAPP clearly

has a diverse and interesting folding landscape. The landscape must contain multiple folding

barriers to cause the different dynamics. Our previous study of unlabeled hIAPP (Sec. 5.4)
 138

suggested a much simpler landscape, since the data could be largely explained with only an

initial random coil state and a final folded state (8). In principle, the shape and width of the

unlabeled β-sheet transition should reflect the different dynamics since it contains the kinetics of

all residues. However, since the unlabeled feature is a convolution of 37 amino acids, the

kinetics of 1 or a few residues with unique transition times will be obscured by the others. These

experiments demonstrate that when individual residues are monitored using isotope labeling,

details of the folding and assembly process are resealed that are otherwise masked by

overlapping spectra features.

As mentioned above, the quantitative determination of all t50 times was done using 2D

data sets smoothed with a 1 min. running average. For visualization purposes, the reported 2D

IR spectra (Figs. 5.6, 5.9 and 5.10) are smoothed with 9 min. windows and the kinetic traces use

an 18 min. window. Nonetheless, to understand the effects of smoothing on our results, we have

also calculated the averages and errors in t50 following the above procedure using various

smoothing windows. Shown in Fig. 5.13 is the labeled t50 data reported in Figs. 5.8B and 5.12D

that was calculated with 1 min. windows compared to the same data calculated from data

smoothed with 18 min. windows. Smoothing with 18 min. windows reduces the error bars and

tends to decrease the variation in the labeled t50 times (as would be expected since the time-

resolution is poorer), but scarcely alter the relative t50 times of the 6 labels. Therefore, we

conclude that reasonable amounts of smoothing do not alter our fundamental conclusions and we

have chosen 1 min. windows for data analysis, even though they give larger error bars, because it

is the more conservative choice.

 139

Figure 5.9 Difference 2D IR spectra at the last stage of aggregation of the six peptides

labeled at Ala8(A), Ala13(B), Val17(C), Ala25(D), Leu27(E), and Val32(F). Each difference

2D IR spectrum was generated by subtracting the spectrum collected at the end of the reaction

from the first 2D IR spectrum of each experiment. Fig S1D is identical to Fig. 2D in the text.

Except for Ala 25, all the other five labeled peptides exhibit a single label feature (black arrows)

around 1580 cm-1 with their frequencies and lineshapes varying as their location in the final fibril

structure. Cross-peaks between the isotope labels and the unlabeled β-sheet peak at 1618 cm-1

are seen in all six labeled peptides (red arrows), indicating strong coupling of the label to the β-

sheets.

1700
1680 A B C
A8 A13 V17
ωpump (c m-1)

1660

1640
1620

1600
1580

1560
1680 D E F
A25 L27 V32
ωpump (c m-1)

1660

1640
1620

1600
1580

1560
1560 1600 1640 1680 1560 1600 1640 1680 1560 1600 1640 1680
ωprobe -1
(c m ) ωprobe -1
(c m ) ωprobe -1
(c m )
 140

Figure 5.10 Representative difference 2D IR spectra at various stages of aggregation of

the six peptides labeled at Ala8(A), Ala13(B), Val17(C), Ala25(D), Leu27(E), and Val32(F).

The center column displays spectra at t = ~t50 of the label. The left column shows spectra at t <

t50 of the label, where as the right column was chosen at t > t50 of the label when the label

intensities begin to plateau. At the top-left corner of each spectrum, corresponding scaled time is

denoted by multiples of t50 of the unlabeled β-sheet. Each difference 2D IR spectrum was

generated by subtracting the spectrum collected at certain time from the first 2D IR spectrum of

each experiment.
 141

0.9t50 1.2t50 1.9t50

A 1680
ωpump (c m -1)

1640
A8
1600

1560
0.6t50 1.1t50 1.6t50
B 1680
ω pump (c m -1)

1640
A13
1600

1560
0.7t50 0.9t50 1.1t50
C 1680
ω pump (c m -1)

1640
V17
1600

1560
0.7t50 0.9t50 1.5t50
D 1680
ω pump (c m -1)

1640
A25
1600

1560
0.6t50 1.1t50 1.6t50
E 1680
ωpump (c m-1)

1640
L27
1600

1560
0.9t50 1.8t50 4.2t50
F 1680
ωpump (c m -1)

1640
V32
1600

1560
1560 1600 1640 1680 1560 1600 1640 1680 1560 1600 1640 1680
ωprobe (c m -1) ωprobe (c m -1) ωprobe (c m-1)
 142

Figure 5.11 Time course of the spectral features (black line) and their fits (red line) from

the best data of six labeled peptides at Ala8(A), Ala13(B), Val17(C), Ala25(D), Leu27(E), and

Val32(F). For each residue, the feature associated with unlabeled b-strand (left column) and the
13 18
C O label (right column) are shown. For visualization purpose, the data and fitted curves are

smoothed with 18 mins window.

β-strand Label β-strand Label

A 160 8 D 160
12
intensity (a.u.)

intensity (a.u.)

intensity (a.u.)

intensity (a.u.)
A8 120 6 A25 120
8
80 4 80
4
40 data 2 data 40 data data
fit fit fit fit
0 0 0
0
40 80 120 160 200 40 80 120 160 200 50 100 150 200 250 50 100 150 200 250
time (min) time (min) time (min) time (min)
B 300
8
E 400
intensity (a.u.)

intensity (a.u.)

intensity (a.u.)

intensity (a.u.)
A13 6
L27 300
30
200
4 20
200
100 2
data data 100 data 10 data
fit fit fit fit
0
0 0
50 100 150 200 50 100 150 200 40 80 120 160 200 40 80 120 160 200
time (min) time (min) time (min) time (min)
C 200 40 F 160 40
intensity (a.u.)

intensity (a.u.)

intensity (a.u.)

intensity (a.u.)
V17 150 30 V32 120 30

100 20 80 20

50 data 10 data 40 data 10 data

fit fit fit fit
0 0 0 0
50 100 150 200 250 50 100 150 200 250 20 40 60 80 100 20 40 60 80 100
time (min) time (min) time (min) time (min)
 143

Figure 5.12 Summary of the kinetics for all 6 labeled peptides. (A) Scaled kinetic curves

for the unlabeled β-sheet feature at 1617 cm-1 for all 6 labeled samples plus an unlabeled peptide

sample obtained from the best experiment among 2 to 4 sets for each peptide. (B) Using the

same scaling and data set, comparison of kinetic curves for the isotope labeled peaks. (C) Ratio

of t50 of the label to t50 of the unlabeled β-sheet for the six residues measured with standard error

bars from the best 2 to 4 data sets. Blue circles represent the best experiment, used for in (A) and

(B), for each labeled peptide. Red, magenta and green asterisks are from other separate

experiments. (D) Averaged ratio of t50 of the label to t50 of the unlabeled β-sheet for the six

residues. All the data in (C) was used to produce the means and error bars.
normalized intensity

100
A B
80

60 A8
A13 A8
40 V17 A13
A25 V17
20 L27 A25
V32 L27
0 UL V32
0.2 0.6 1.0 1.4 1.8 0.2 0.6 1.0 1.4 1.8
normalized time (t/t50) normalized time (t/t50)
t50(label) / t50(unlabeled)

1.6
C D
1.4

1.2

1.0

0.8
5 10 15 20 25 30 35 10 15 20 25 30 35
residue number residue number
 144
13
Figure 5.13 Effects of running average in the ratio of t50 between C18O label and the

unlabeled 12C16O for (A) four separate experiments on the same peptide labeled at Ala 25 and (B)

six separate experiments on six peptides labeled at six different residues. β-strand and label

kinetics from data generated in two different ways were separately fitted with Eq. S1 to generate

the ratio. Blue circles are from data averaged with 1 min window and red asterisks are from data

smoothed with 18 min window. Standard error bars are shown.

1.2 1.6
t50 of label / t50 of unlabeled

t50 of label / t50 of unlabeled

A 18 min window B 18 min window
1 min window 1 min window
1.1 1.4

1.0 1.2

0.9 1.0

0.8 0.8
1 2 3 4 5 10 15 20 25 30 35
Repeats of the experiment residue number
 145

5.5.5 Peak positions and intensities of isotope-labels in 2D IR spectra

The above experiments reports on local structure through the changes in intensity and

frequency of the isotope labeled amide I band. There are two factors that affect the frequency

and intensity the bands in the 2D IR spectra: their environment and their coupling. The

electrostatic environment around the atoms of the amide I mode (the backbone C, O, N, and H

atoms) influence its frequency. Electrostatics is largely influenced by solvation and hydrogen

bonding, which is why the amide I band has a lower frequency in water than in membrane bound

systems. As for coupling, the isotope labeled band is vibrationally coupled to nearby residues,

which can also shift its frequency. These couplings create the cross peak to the unlabeled amide
13 18
I mode. However, the coupling to other C O labels is more important for the observed

frequency, because the degeneracy of the modes leads to larger frequency shifts. To ascertain

the effects of electrostatics versus coupling, we collected 2D IR spectra of Ala13, Ala25 and

Leu27 where only 25% of the peptides contain an isotope label, thereby largely eliminating the

effects of coupling between labeled residues. (The spectra are reported in Fig. 5.10) We found

that upon isotope dilution, the frequencies of these three residues lie within 5 cm-1 of each other

(1589 to 1594 cm-1), indicating that their local electrostatics are comparable. Moreover, the

isotope frequencies are higher when diluted, indicating that the coupling between isotope labels

causes a negative frequency shifts, which is consistent with the negative coupling constants

predicted for β-sheets in amyloid fibril (26). These results emphasize that the isotope labeled

features that are the focus of this study arise from delocalized vibrational modes that involve the

cooperative motions of several labeled amide I groups. Thus, the kinetics experiments are not

monitoring the folding of a single peptide, but are instead measuring β-sheet formation through

the association of multiple peptides into columns of isotopically labeled residues.

 146

Figure 5.14 Diagonal slices of 2D IR spectra taken for matured fibers of three peptides

labeled at Ala13(A), Ala25(B), and Leu27(C) in a 100% (solid) and 25% (dashed) concentration

of the isotope.
normalized intensity (a.u.)

20 A B C
A13 A25 L27
15 A13/UL A25/UL L27/UL

10

0
1570 1580 1590 1570 1580 1590 1570 1580 1590 1600
frequency (cm-1) frequency (cm-1) frequency (cm-1)
 147

5.6 Discussion

Our data is consistent with the mechanism shown in Fig. 5.15 whereby a series of peptides

nucleate into a β-sheet conformation at Val17 on the N-terminal end of the ordered loop,

followed by the formation of the ordered loop to nucleate a second β-sheet at Ala25 on the C-

terminal end of the loop. The N-terminal β-strand then folds about twice as quickly as the C-

terminal strand. Some portion of the ensemble may be nucleating at Ala25 before Val17, since

the kinetic trace of Ala25 follows closely enough behind Val17 that the two kinetic curves

partially overlap (Fig. 5.13B). Also broadly consistent with the data would be nucleation at the

ordered loop, followed by folding down the N- and C-terminal β-strands. We do not yet have a

spectroscopic signature for when loop formation occurs or independent measures for each of the

two columns, but we suspect that the two columns of peptides grow at different rates. Different

growth rates would explain why the C-terminal sheet, which resides at the interface of the two

columns (see Fig. 5.5B), has slower kinetics than the outside N-terminal sheet e.g. the region

containing Val32 does not form β-sheet until it is stabilized by the second column of peptides.

In principle, more sophisticated isotope labeling schemes, like using pairs of labels that span

across the β-strands, could be used to test this hypothesis. Regardless, it is clear that nucleation

occurs at or near Val17, followed by elongation of the two parallel β-strands, with the N-

terminal β-strand forming more rapidly. The folding mechanism is somewhat reminiscent of the

zipper mechanism for β-hairpin folding. In the zipper mechanism, the β-turn forms first,

followed by elongation of the hairpin towards the N- and C-termini by rapidly forming a series

of hydrogen bonds (30, 31). However, we emphasize that the hydrogen bonding in amyloid is

not the same; instead of hydrogen bonds linking the two halves of each peptide individually,
 148

hydrogen bonds in amyloids run along the fiber long axis to connect stacked hairpins. Thus,

fundamentally different forces are likely to be responsible for hairpin formation in amyloid.

Another very important point is that our data rules out the presence of large β-sheet structures

during the lag phase. We estimate that not more than 5% of the peptide ensemble can reside in

β-sheets with more than 4 strands, based on the background intensity at 1618 cm-1 where large β-

sheets absorb. β-sheet aggregates of 3 strands or less could be present during the lag phase,

because small β-sheets absorb near the random coil at 1645 cm-1 (32).

Of the six residues measured, Ala25 is particularly interesting because it is the only

substitution that exhibits two isotope labeled bands (See Figs. 5.6B to D for Ala25 and SI Figs.

5.9 and 5.10 for other residues). Isotope dilution of Ala25 causes both peaks to collapse into a

single feature (Fig. 5.14). This finding indicates that Ala25 contains two coupling constants of

3.5 and 15.5 cm-1. Two coupling constants indicate that there is a bimodal distribution of

distances between adjacent Ala25 residues. A bimodal distribution of distances could arise from

two different populations of fibril structures or from the helix twist which breaks the symmetry

between the two columns of peptides. The last possibility seems unlikely, since the twist is small

over the delocalization length of a vibrational exciton (4-6 strands), and thus should be negligible.

It seems more likely that there are two subpopulations of β-sheet structures and that the relative

ratio of the 1574 and 1585 cm-1 features gives their relative population. If true, then the fact that

the two features have similar intensities up to 1.5t50 indicates that the two subpopulations

aggregate in tandem, but that the subpopulation with the lower frequency isotope label (and, thus,

shorter Ala25 spacing) is eventually preferred (Fig. 5.7A). Considering that Ala25 is the only

residue that exhibits two peaks and the only residue we measured that was assigned to random
 149

coil in the ssNMR structure, it appears that the ordered loop is the most sensitive region of

the structure to this bimodal distribution.

At this time, we do not know why nucleation occurs near Val17. However, some insight

may be gained by considering our results in the context of the well known correlation between

variations in the primarily sequence of IAPP and the prevalence of islet amyloid in different

species (20). For example, rodents do not form islet amyloid even though they produce IAPP.

The sequence of rat IAPP differs from that of hIAPP at six positions; residues 18, 23, 25, 26, 28

and 29. Four of these residues flank the ordered loop and are located close to the amino acids

that we postulate are the nucleation sites for β-sheet formation. It is also worth noting that

modification of residues 24 and 26 by N-methylation leads to a non amyloidogenic variant of

human IAPP as does a single mutation from Ile26 to proline (33, 34). Interestingly substitutions

outside of the 20-29 region but within the region we have identified as being involved with

nucleation also profoundly affected amyloid formation. A triple mutant in which residue 17, 19

and 30 were replaced by proline had a dramatically reduced tendency to aggregate (35). Thus

studies all highlight the putative importance of the region(s) we have identified as being critical

for nucleation. Our results have important implications for inhibitor development since they

provide the information required to design inhibitors which target the critical nucleation site.

Along these lines insulin and its isolated B-chain are known to be among the most potent

inhibitors of amyloid formation by IAPP and they are thought to interact predominately within

residues 7 to 19 of IAPP (36). Thus, rat and mouse IAPP might have evolved to prohibit

amyloid formation by mutating the first two sites in the aggregation pathway: Val17 and Ala25.

In principle, much of the information reported here could have been obtained using FTIR

spectroscopy. However, we have not succeeded at resolving the kinetics of the isotope labels in
 150

these samples using FTIR spectroscopy. One advantage of 2D IR spectroscopy over FTIR is

that background signal from weak absorbers is suppressed because the intensity of 2D IR spectra

scales as |µ|4, where µ is the transition dipole, whereas FTIR scales as |µ|2. Thus, the non-linear

nature of 2D IR spectroscopy enhances strong absorbers. Another advantage of our rapid-scan

technology for collecting 2D IR spectra is that we can phase-cycle the pulses to help eliminate

scatter from these heterogeneous samples. But the real advantages of 2D IR spectroscopy will be

utilized in the future in experiments that monitor 2D lineshapes and quantify couplings which

have the potential to time-resolve solvent expulsion during fiber formation and identify the

secondary structures of intermediates.

 151

Figure 5.15 hIAPP aggregation pathway that is consistent with our data. Labeled residues

in register at each assembly stage are denoted with their residue numbers and colored circles

(purple: Val17, red: Ala25, cyan: Leu27, blue: Ala13, yellow: Ala8, green: Val32). Thick black

arrows are β-strands, while the portion of the peptide in random coil or loop is shown in gray.
 152

5.7 Conclusion

We have utilized isotope labeling and new technological advances in 2D IR spectroscopy to gain

residue specific kinetic information about the development of the aggregation pathway of hIAPP.

We found that the amyloid fibrils associated with type 2 diabetes follow a multistep pathway

involving a series of intermediate, largely β-sheet structures. The methodology presented here is

not limited to IAPP nor is it limited to amyloid formation in homogenous solution. Membrane

peptide interactions have been postulated to play an important role in amyloid formation (37) and

our methodology is well suited to investigate such systems. It is also applicable to a wide range

of other proteins now that advances in peptide synthesis have made it possible to synthesize large

polypeptides with unnatural or labeled amino acids (38). Furthermore, 2D IR spectra can be

simulated from X-ray, NMR or molecular dynamics structures. Thus, our methodology has the

potential to test and link disparate studies that together hold the promise of providing a

comprehensive understanding of the elusive mechanism of amyloid fiber formation.

5.8 Acknowledgments

We thank Professor Daniel Raleigh and Dr. Ruchi Gupta in State University of New

York at Stony Brook for the collaboration and for providing the synthesized peptides.
 153

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 157

Appendix I

List of Publication

11. Ling YL, Strasfeld DB, Shim S-H, Raleigh DP, and Zanni MT, “2D IR provides evidence of
an on-pathway intermediate in the membrane-catalyzed assembly of diabetic amyloid”,
Submitted.

10. Shim S-H and Zanni MT, “How to turn your pump-probe instrument into a multidimensional
spectrometer: 2D IR and Vis spectroscopies via pulse shaping.” Invited article for Phys.
Chem. Chem. Phys. Accepted.

9. Shim S-H, Gupta R, Ling YL, Strasfeld DB, Raleigh DP and Zanni MT, “2D IR
spectroscopy defines the pathway of amyloid formation with residue specific resolution.”
Submitted.

8. Xiong W, Strasfeld DB, Shim S-H, and Zanni MT (2008) “Automated 2D IR spectrometer
mitigates the influence of high optical densities.” Vibrational Spectroscopy, Accepted.

7. Strasfeld DB, Ling YL, Shim S-H, and Zanni MT (2008) “Tracking fibril formation in
human Islet amyloid polypeptide with automated 2D-IR spectroscopy.” J. Am. Chem. Soc.
130:6698-6699.

6. Grumstrup EM,* Shim S-H,* Montgomery MA,* Damrauer NH, & Zanni MT (2007)
“Facile collection of two-dimensional electronic spectra using femtosecond pulse-shaping
technology.” Optics Express 15(25):16681-16689. *Equal authors.

5. Shim S-H, Strasfeld DB, Ling YL, & Zanni MT (2007) “Automated 2D IR spectroscopy
using a mid-IR pulse shaper and application of this technology to the human islet amyloid
polypeptide.” Proc. atl. Acad. Sci. U.S.A. 104:14197-14202.

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1. Shim S-H,* Strasfeld DB,* Fulmer EC, & Zanni MT (2006) “Femtosecond pulse shaping
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