Up-Close and Personal Investigation of Beta-Carotene)

Home , Isoprene, Peridinin

Single Molecule Study of Beta-Carotene using Scanning Tunneling Microscope

(Up-close and Personal Investigation of Beta-Carotene)

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Timur Skeini

June 2010

This dissertation titled

Single Molecule Study of Beta-Carotene using Scanning Tunneling Microscope

(Up-close and Personal Investigation of Beta-Carotene)

TIMUR SKEINI

has been approved for

the Department of Physics and Astronomy

and the College of Arts and Sciences by

Saw W. Hla

Associate Professor of Physics and Astronomy

Benjamin M. Ogles

Dean, College of Arts and Sciences 3

ABSTRACT

SKEINI, TIMUR, Ph.D., June 2010, Physics and Astronomy

Single Molecule Study of Beta-Carotene using Scanning Tunneling Microscope

(Up-close and Personal Investigation of Beta-Carotene) (101 pp.)

Director of Dissertation: Saw W. Hla

We mimic beta-carotene (β-carotene) environment in plant leaves by using

Au(111) as a substrate and conduct single molecule level studies in ultra high vacuum at low substrate temperatures of 77 K and 4.2 K. The STM images and manipulations studies identify five mechanically stable beta-carotene conformations on this substrate, with two never before reported by experiments. We confirm beta-carotene single molecule switching and molecular wire functionality. The beta-carotene clusters are disordered and rich in conformations. Single molecule extraction and cluster manipulation experiments indicate that intermolecular interactions of beta-carotene on

Au(111) are stronger than molecule-substrate interactions. The mixtures of beta-carotene and chlorophyll-a result in three regions of ordered self-assembled chlorophyll-a, mobile beta-carotene, and their disordered mixtures with a ratio of 43.6±7.8% to 56.4±10.1%

(over 9 mixtures), respectively, with 56.0±10.1% of beta-carotene being trans, indicating beta-carotene preferential mixing with chlorophyll-a instead of its own beta-carotene clusters. The results of this dissertation provide information on single molecule level properties of beta-carotene for fundamental understanding as well as for applications in green nano-bio-technology, nano-medicine, molecular electronics, and energy harvesting.

Approved: ______

Saw W. Hla

Associate Professor of Physics and Astronomy 4

ACKNOWLEDGMENTS

- Advisor Dr. Saw Wai Hla, Group and Involved Students

www.phy.ohiou.edu/~hla/

- Dissertation Committee:

Dr. Ido Braslavsky, Dr. Alexander Neiman, Dr. Hugh H. Richardson

- Department of Physics and Astronomy, Faculty, Staff, Students

(Dr. Alexander Govorov, Dr. David F. J. Tees, Dr. Sergio Ulloa, Ennice Swiegart,

Tracy Inman and MANY others), Department of Chemistry, ISFS, and others

- Ohio University:

Condensed Matter Surface Science (CMSS),

Quantitative Biology Institute (QBI),

Nanoscale Quantum Phenomena Institute (NQPI)

- Financial Support:

Department Physics and Astronomy, Ohio University;

USA Department of Energy (DOE) DE-FG02-02ER46012;

National Science Foundation (NSF).

- All others who helped in whatever way with my research, this work and for my PhD.

- Thank you to the Universe, Life and Everything!  5

DEDICATION

TABLE OF CONTENTS Page

Abstract ...... 3 Acknowledgments...... 4 Dedication ...... 5 List of Tables ...... 8 List of Figures ...... 9 Chapter I: Introduction ...... 11 1.1 Background and Motivation ...... 11 1.2 Overview ...... 14 Chapter II: Instrumentation and Experimental Techniques ...... 17 2.1. How an STM Works ...... 17 2.2. Lateral Manipulation ...... 20 2.3. Electron Induced Manipulation ...... 22 Chapter III: Background Materials: Molecules of Current Study and the Substrate ...... 24 3.0. Introduction ...... 24 3.1. Beta-Carotene (β-carotene) ...... 26 3.2. Chlorophyll-a ...... 32 3.3. Au(111) Substrate ...... 33 3.4. Molecular Deposition ...... 33 Chapter IV: Single Molecule Studies ...... 35 4.1. Stable Beta-Carotene Conformations on Au(111) ...... 36 4.1.1 Trans Conformation ...... 36 4.1.2 Cis Conformation ...... 38 4.1.3 Twist Conformation ...... 39 4.1.4 V-Shape Conformation ...... 40 4.1.5 V-Twist Conformation ...... 42 4.2. Mechanical Properties of Single Molecules ...... 43 4.2.1 Trans Conformation ...... 44 4.2.2 Cis Conformation ...... 45 4.2.3 Twist Conformation ...... 46 7

4.2.4 V-Shape Conformation ...... 47 4.2.5 V-Twist Conformation ...... 48 Chapter V: Single Molecule Conformation Switching ...... 51 5.1. Cis-Trans Isomerization using Tunneling Electrons ...... 51 5.2. Cis-Trans Isomerization using Force ...... 52 5.3. V-Twist Conformation Switching ...... 54 5.4. Conformation Switching of End Pi-Ring ...... 55 5.5. Single Molecule Dynamics ...... 57 5.6. Direct Evidence of Beta-Carotene as a Molecular Wire ...... 59 Chapter VI: Molecular Assembly – Beta-Carotene Clusters on Au(111) ...... 61 6.1. Single Molecule Extraction ...... 63 6.2. Beta-Carotene Clusters ...... 69 6.3. Cluster Manipulation ...... 74 Chapter VII: Beta-Carotene – Chlorophyll-a Complexes ...... 76 7.1. Previous STM Experiments ...... 76 7.2. Our Results ...... 76 Chapter VIII: Conclusion ...... 82 8.1. Beta-Carotene ...... 82 8.2. Beta-Carotene and Chlorophyll-a ...... 84 Appendix A: Cluster Composition Statistics (Beta-Carotene) ...... 86 Appendix B: Mixture Composition Statistics (Beta-Carotene + Chlorophyll-a) ...... 88 Appendix C: Manipulation Statistics ...... 92 References ...... 94

LIST OF TABLES

Page

Table I: Cluster percentage composition comparison with literature ...... 73 9

LIST OF FIGURES

Page

Fig. 2.1. STM schematic. LT-UHV-STM at Dr. Hla lab photo (Ohio University) ...... 18 Fig. 2.2. Lateral Manipulation (LM) schematic...... 21 Fig. 2.3. Lateral manipulation modes with STM tip-height curves...... 21 Fig. 2.4. Electron Induced Manipulation ...... 23 Fig. 3.0. Abudance of beta-carotene in photosynthesis ...... 24 Fig. 3.1. Structure of the Photosystem II...... 25 Fig. 3.2. Constitution formulas of s-cis and all-trans-beta-carotene ...... 27 Fig. 3.3. Thiol-substituted carotenoids ...... 27 Fig. 3.4. beta-carotene model systems ...... 28 Fig. 3.5. Conformations of beta-carotene that were observed and others ...... 30 Fig. 3.6. Relative energies of beta-carotene isomers and dihedral angles ...... 31 Fig. 3.7. Chemical structure of chlorophyll-a ...... 33 Fig. 3.8. Schematic of used home built Knudson cell ...... 34 Fig. 4.0. Five mechanically stable conformations of beta-carotene determined in this study ...... 35 Fig. 4.1. Trans conformation ...... 37 Fig. 4.2. Cis conformation ...... 38 Fig. 4.3. Twist conformation ...... 40 Fig. 4.4. V-Shape conformation ...... 41 Fig. 4.5. V-Twist conformation ...... 43 Fig. 4.6. Proof of stability for trans conformation ...... 45 Fig. 4.7. Proof of stability for cis conformation ...... 46 Fig. 4.8. Proof of stability for twist conformation ...... 47 Fig. 4.9. Proof of stability for v-shape conformation ...... 48 Fig. 4.10. Proof of stability for v-twist conformation ...... 49 Fig. 4.11. V-Twist rotation lateral manipulation signal explanation ...... 50 Fig. 5.1. Cis-Trans switching using tunneling electrons ...... 52 Fig. 5.2. Cis-Trans switching using lateral manipulation ...... 53 Fig. 5.3. V-Twist Conformation Switching ...... 55 Fig. 5.4. Conformation switching of end pi-ring ...... 57 Fig. 5.5. Single molecule dynamics of two molecules pulled apart ...... 58 Fig. 5.6. Evidence of beta-carotene as a molecular wire ...... 60 Fig. 6.0. Beta-carotene clusters on Au(111) overview ...... 61 Fig. 6.1. Analysis of partial pullout of cis conformation from molecular cluster ...... 66 10

Fig. 6.2. The full molecule pullout lateral manipulation sequence ...... 67 and 68 Fig. 6.2.0. Beta-carotene clusters overview of Au(111) ...... 69 Fig. 6.3. Composition analysis of two typical beta-carotene clusters ...... 70 Fig. 6.4. Cluster composition statistics ...... 71 Fig. 6.5. Percent composition trends for beta-carotene isomers ...... 74 Fig. 6.6. Cluster manipulation using Lateral Manipulation technique ...... 75 Fig. 7.1. STM topography images of the chlorophyll-a and beta-carotene mixture ...... 78 Fig. 7.2. Content of analysis of beta-carotene and chlorophyll-a mixture ...... 80 Fig. 7.3. Content percent composition of chlorophyll-a and beta-carotene mixture ...... 80 Fig. 7.4. Content percent composition of chlorophyll-a within the mixtures ...... 81 Fig. 7.5. Content percent composition of beta-carotene within the mixtures ...... 81 Fig. A.1. Appendix A: Cluster composition statistics ...... 86 Fig. A.2. Appendix A: Beta-carotene extra clusters 2 to 8 (except cluster 5 and 6) ...... 87 Fig. A.3. Appendix A: Beta-carotene extra clusters 9 to 11 ...... 87 Fig. B.1. Appendix B: Composition statistics for beta-carotene and chlorophyll-a mixtures ...... 88 Fig. B.2. Appendix B: Beta-carotene and chlorophyll-a extra mixtures 2 and 3...... 89 Fig. B.3. Appendix B: Beta-carotene and chlorophyll-a extra mixtures 4 and 5...... 89 Fig. B.4. Appendix B: Beta-carotene and chlorophyll-a extra mixture 6 ...... 90 Fig. B.5. Appendix B: Beta-carotene and chlorophyll-a extra mixtures 7 and 8...... 90 Fig. B.6. Appendix B: Beta-carotene and chlorophyll-a extra mixture 9 ...... 91 Fig. C.1. Appendix C: Manipulations presented in this dissertation ...... 92 Fig. C.2. Appendix C: Count of STM data files relevant to this dissertation ...... 93

CHAPTER I: INTRODUCTION

1.1 Background and Motivation

The main thesis (aim) of this dissertation, as foreshadowed in the abstract, is to help facilitate the much demanded solutions to the vital problems of current civilization in energy, electronics, and medicine, and we do this by providing the fundamental scientific research in the growing field of now-realizable nano-bio-technology, that we think is capable in solving these problems, with a specific focus on advancing “green” technologies toward the bottom up assembly of bio-nano-devices.

The followings will elaborate on these concepts and on how do we address these aims.

The society currently needs "green" devices mainly for the energy, electronic and medical applications. One solution is to build nanoscale biomolecular devices. This requires fundamental scientific understanding of nanoscale biomolecules. Thus in this dissertation, we study and manipulate the chemical and physical properties of biodegradable plant photosensitive biomolecules, such as, beta-carotene, and chlorophyll- a, on molecular level using the scanning tunneling microscope (STM).

A "green" device is manufactured in an environmental-friendly way from biologically non-toxic components commonly found in plants and usually easily disposable

(biodegradable) [1-2]. Similarly "green" energy is obtained in environmental-friendly 12 way resulting in no adverse environmental conditions and producing no pollution, non- toxic biodegradable output, or output that can be broken down or converted to non-toxic biodegradable components. Examples include solar and wind power, plant and hydrogen fuels.

The demand exists due to the decreasing mainstream energy resources, such as, fossil fuels and oil, as well as, the growing number of medical conditions and environmental disasters due to the growing toxicity in pollution, caused by the growing consumption of energy, the growing use of the toxic device components and the associated environmentally-adverse manufacturing processes [1-2].

Our choice of molecules addresses some of the above issues of alternative energy sources and green devices, improving medical applications and reducing pollution. These molecules are environmental-friendly in the ways that they are biologically non-toxic, easily available, easily extracted with no environmentally-adverse effects and are biodegradable. They all occur in living tissues: chlorophyll-a and beta-carotene are mainly obtained from plants [3-5].

One of the popular and pollution-free ways to get "green" energy is to harvest light.

Pigment porphyrin molecules, like chlorophyll-a, and beta-carotene, could potentially be integrated into solar cells due to their photosensitive nature [3-5]. Experimental photo- detection systems already show promising results [6]. The fundamental understanding of 13 natural photosynthesis, key in the evolution of life [7], might allow the construction of the artificial photosynthetic systems with much higher efficiency than existing solar cells

[3,5,7-13].

These molecules also have important medical potentials. Beta-carotene is a carotenoid – anti-oxidant, that may prevent cancer cell damage [14]. It is also vitamin B12, a precursor of vitamin A - essential in human vision [15]. Isomerization of retinal molecule is the heart of human vision [16] and photo-detection [6,17], and impacts medical conditions like Childhood Blindness, Stargardt Macular Dystrophy, Leber

Congenital Amaurosis [16,18]. Beta-carotene together with retinal can also improve the stability and absorption range of a system [19]. The fundamental understanding of these molecules has strong impact on future medical vision implants, probes, artificial organs and treatments.

In the nanotechnology front, the potential of molecular electronic devices is becoming comparable to semiconductor electronics in memory storage [6,20], circuit logic [21], voltage sensors [22] and components [23]. Molecular self-assembly is a major advantage in molecular electronics [21,24] and the key in bottom-up nanoscience [12,13].

Switchable conformations, fast isomerization and understanding of charge transfer in chlorophyll-a [25], beta-carotene [12] and retinal [17] might yield bio-transistors and biological circuits. Beta-carotene can also be used as molecular wire [12,26]. The intrinsic photosensitive nature of these pigment molecules allows construction of molecular 14 optoelectronic devices like "green" solar cells [27], retinal photo-detectors [6], hybrid/cybernetic devices for artificial vision, fiber optic connectivity and optical circuitry.

1.2 Overview

Chapter I introduces the topics and goals involved, as background information and motivation, and provides an overview of the dissertation.

Chapter II introduces the instrumentation used, the Low Temperature Ultra High Vacuum

Scanning Tunneling Microscopy (LT-UHV-STM), and the experimental techniques employed in this work, such as, the Lateral Manipulation (LM) and the Electron Induced manipulation.

Chapter III provides background information on materials used for this work, the beta- carotene and chlorophyll-a molecules and the Au(111) substrate, and describes the details of depositing the molecules on the substrate.

Chapter IV focuses on the single molecule studies specifically identifying the stable beta- carotene conformations on Au(111) by presenting the observed STM topography images for each conformation (Trans, Cis, Twist, V-Shape and V-Twist). The corresponding chemical formulas and Au(111) lattice adsorption sites, and evidence of mechanical stability for each conformation are presented. Here, lateral manipulation or induced 15 tunneling excitation sequence result in lateral translation of the molecule and/or rotation about adsorption sites by preserving the initial molecule conformation.

Chapter V is devoted to the single molecule conformation switching. It discusses Cis-

Trans isomerization using tunneling electrons and force, V-Twist conformation switching, and the conformation switching of end pi-ring of beta-carotene. It also discusses in detail the dynamics of single molecules as determined from the manipulation signals and provides a direct evidence that beta-carotene can act as a molecular wire.

Chapter VI discusses the molecular assembly of beta-carotene in clusters on Au(111) surface. Here, we illustrate in detail how the single molecules were extracted from the molecular clusters. It also provides the composition statistics of identified stable beta- carotene conformations within the molecular clusters. Finally it presents the lateral manipulation of the entire cluster, showing that its structural integrity is preserved, which indicates that the intermolecular interactions within the cluster are stronger that the molecule-substrate interactions on Au(111) substrate.

Chapter VII presents our experimental observations on the molecular clusters formed by a mixture of chlorophyll-a and beta-carotene with less than Langmuir-Blodgett monolayer coverage on Au(111) substrate. It shows three regions; ordered self- assembled chlorophyll-a region, high intensity (mobile) beta-carotene region, and chlorophyll-a and beta-carotene mixture region. It shows that the mixture regions can be 16 analyzed for single molecule conformations of beta-carotene and chlorophyll-a, which provides compositional statistics.

Finally Chapter VIII concludes this work with a summary of beta-carotene and chlorophyll-a experimental results and following conclusions and implications, such as beta-carotene being a molecular wire can be used for nanoscale devices, and beta- carotene with chlorophyll-a mixture composition being important for understanding photosynthesis. 17

CHAPTER II: INSTRUMENTATION AND EXPERIMENTAL TECHNIQUES

2.1. How an STM Works

The Scanning Tunneling Microscope (STM) is a nanoscale research tool that allows to study surfaces with atomic precision, such as, imaging [1,28], manipulation [1,29] and engineering [1]. It consists of metallic tip connected in a circuit, which is placed close above a conducting surface of study (Fig. 2.1) [30]. When the tip is less than 1 nm from the surface and the voltage (order ~ a few V) between the tip and substrate is applied, then the electrons tunnel between the tip and the substrate. The tunneling current (order of ~1 nA) decreases exponentially as the tip-substrate distance increases [31].

Theoretically this is a 1-dimension quantum mechanical tunneling through a potential barrier [30]. The low temperature (LT) STM used for this dissertation can be operated either in liquid nitrogen (LN2) temperature (~77K), or in liquid helium (LHe) temperatures (~4.2K) with lateral drift of ~ 0.1 nm/hour [31]. The ultrahigh vacuum

(UHV) environment having a pressure below 10-11 Torr results in several weeks of working time on Au(111) surface [31].

Fig. 2.1. (Top) STM schematic [30], (Bottom) LT-UHV-STM at Dr. Hla lab photo (Ohio University).

The STM was invented by Binnig and Rohrer [32] who received a 1986 Nobel prize for it. The first atomic-scale man-made structure constructed with an STM tip was an IBM logo consisting of Xe atoms on Ni surface at 4K [33-34].The lateral resolution of an STM depends on the tip radius R and the vacuum gap distance „d‟ approximately as [2Å

(R+d)]1/2 [35].

The first approximation for tunneling current expression by Fowler-Nordheim [36] is

(Eq. 2.1)

where U is an applied voltage between the tip and the surface, d is their separation, W is the average work function, and K is a constant (typically ~1.025 Å-1 (eV)-1/2 for a vacuum gap).

Using the STM it is possible to obtain a topography image of a conducting surface or an object of study on such surface with atomic precision [1,28]. Using tunneling spectroscopy [1] it is possible to measure physical properties such as electronic structure and vibrational signatures of individual molecules adsorbed on a surface [1,37-39].

Single atoms and molecules can also be recognized, manipulated and be used as building blocks in nano-structures [1,29].

The tip preparation before scanning involves crashing the tip into the sample surface in a controlled way resulting in a single-atom tip [40]. This procedure can also be used to improve the tip after a scan or manipulation, fix a multiple tip and remove adsorbent molecules from the tip-apex.

The Scanning Tunneling Spectroscopy (STS) can be used to study the local electronic structure of the substrate [31]. By controlling the tip position and tip-sample voltage, it is possible to measure the differential conductance, dI/dV, that is proportional to the local density of states (DOS), ρs, of the sample surface when the tip DOS, ρT, are constant, since the tunneling current, I, depends on the tip and sample DOS [35] (Eq. 2.2).

(Eq. 2.2)

The STS measurements are usually performed by turning off the STM feedback thereby the tip height is fixed, and then adding a small AC modulation to the DC bias. The first derivative of the tunneling current signal is directly measured by using a Stanford

Research Systems SR830 Lock-In Amplifier.

2.2. Lateral Manipulation

By controlling the tip-sample interactions it is possible to move atoms or molecules laterally across the surface, namely perform the lateral manipulation procedure (LM)

(Fig. 2.2) [33,41-49]. 21

Fig. 2.2. (Left). Lateral Manipulation (LM) schematic [33,41-45,48]. Fig. 2.3. (Right). Lateral manipulation modes with STM tip-height curves: (1) pulling, (2) pushing, and (3) sliding [42-43,45-46,48-49].

In LM the tip is approached toward the sample and then moved laterally while maintaining the tip-sample interaction. There are three possible LM modes (Fig. 2.3): pulling, pushing and sliding [42-43,45-46,48-49]. In pulling/pushing mode the attractive/repulsive interactions pull/push the atom/molecule along the surface resulting in a saw-tooth current/tip-height vs tip-position curve (Fig. 2.3 (1-2)). When the lateral force component on the manipulated atom/molecule is sufficient to overcome the potential barrier between the next lattice site closest to the direction of manipulation then the atom/molecule will jump to that site. Depending on the orientation of crystal lattice and the direction of motion sometimes it will be more energetically favorable for the atom/molecule to jump through several sites rather than jumping to the next site directly.

In sliding mode the atom/molecule is trapped under tip or bound to tip [41-42] resulting in a smooth current/tip-height vs tip-position curve (Fig. 2.3 (3)). The LM method can be used to construct atomic scale structures [34].

2.3. Electron Induced Manipulation

The molecules can also be manipulated using Inelastic-Electron Tunneling (IET) induced manipulation processes [25,45,49-58] inducing rotational, vibrational, or electronic excitations [51,55] (Fig. 2.4). During the IET procedure the tip is positioned above the molecule and low energy tunneling electrons (or holes in negative bias, tunneling from the surface to the tip) are injected into the molecule (Fig. 2.4 A). When tunneling electron energy is transferred to the molecule through a resonance state, a controlled excitation occurs [59]. In this process, the maximum tunneling electron energy is limited by the applied bias. The excitation rate, R, depends on the tunneling current, I, and the number of tunneling electrons, N, via the relation (Eq.2.3)

(Eq. 2.3)

(Fig. 2.4 B) [50-51]. Both single or multiple excitations are possible [50-51] (Fig. 2.4 C).

Multiple excitations can be explained using a harmonic oscillator (Fig. 2.4 C) model, where the first electron-energy transfer causes excitation to a particular energy level and subsequent electron-energy transfer by additional electrons cause that energy level to increase further. This process requires a longer lifetime to couple excitations.

Fig. 2.4. Electron Induced Manipulation. (A) Model showing that during the IET procedure the tip is positioned above the molecule and tunneling electrons (or holes) are injected into the molecule producing an excitation [59]. (B) The excitation rate, R, depends on the tunneling current, I, and the number of tunneling electrons, N [50-51]. (C) Both single or multiple excitations are possible, with multiple excitations explained using a harmonic oscillator model [50-51].

CHAPTER III: BACKGROUND MATERIALS:

MOLECULES OF CURRENT STUDY AND THE SUBSTRATE

Fig. 3.0. A picture drawn by the author indicating abudance of beta-carotene in photosynthesis, which can be concluded from the fact that as a result of changes in the photosynthesis during autumn season the leaves change to colors corresponding with the spectral properties of plant molecules including beta-carotene and chlorophyll-a [2,60-62].

3.0. Introduction

The molecules beta-carotene and chlorophyll-a are involved in the photosynthesis in the oxygenic and non-oxygenic systems, such as, Photosystem I (PS1) and Photosystem II

(PS2) [60-67]. In PS2, beta-carotene has been found in the reaction center and antenna complexes [60] (Fig. 3.0). Photosynthesis studies suggest that beta-carotene in addition to 25 functioning as an antioxidant [66-67] also covers a region of the visible spectrum not accessible by chlorophyll [63], as a result of and/or in addition to suppressing excess light

[60,63] and preventing photodegradation of chlorophyll-a [60,65] (Fig. 3.0). The crystal structures of the various photosynthetic complexes involving chlorophyll and various carotenoids (including beta-carotene) have been reported [64,68], such as, Photosystem II

(PS2) core complexes from Thermosynechococcus elongates, Light-harvesting complex 2

(LHC2) from Rhodospirillum molischianum, Peridinin Chlorophyll-a Protein (PCP) from

Amphidinium carterae, and Photosystem I (PS1) from Thermosynechococcus elongates.

The structural studies of these systems show that both chlorophyll and beta-carotene present in these systems (Fig. 3.1) [61-62,64].

Fig. 3.1. Structure of the PS2. View along the membrane normal of the redox cofactors involved in the secondary electron transfer reactions with arrows denoting steps involving ChlZ, Cyt b559 and beta-carotene [61-62].

In medicine, the health benefits from the dietary intake of (antioxidant) beta-carotene

(and lutein) / chlorophyll has been reported [69]. In photosynthesis beta-carotene covers a region of the visible spectrum not accessible by chlorophyll and protects against excessive light [63]. Pigment porphyrin molecules, obtained mainly from plants and bacteria, like beta-carotene, and chlorophyll-a, could thus be potentially integrated into solar cells due to their photosensitive nature [3-5,17]. Experimental photo-detection systems already show promising results [6]. The fundamental understanding of natural photosynthesis, key in the evolution of life [7], might allow the construction of the artificial photosynthetic systems with much higher efficiency than existing solar cells

[3,5,7-13].

The intrinsic photosensitive nature of these pigment molecules allows construction of molecular optoelectronic devices like "green" solar cells [27], retinal photo-detectors [6], hybrid/cybernetic devices for artificial vision, fiber optic connectivity and optical circuitry.

3.1. Beta-Carotene (β-carotene)

Chemically beta-carotene (β-carotene) is a hydrocarbon derivative of carotenoids (C40), sub-classified from terpenoids (terpenes or isoprenoids) according to the number of isoprene units, and consists of a polyene chain with nine conjugated double bonds and two b-ionone rings [70]. Its chemical formula is C40H56, and has a molecular weight of

536.87 amu. In nature, the beta-carotene appears as two main configuration isomers; the all-trans and the natural abundant (C6-C7)-s-cis forms (Fig. 3.2) [64,70]. Other 27 structurally related carotenoids in photosynthesis are spheroidene, rhodopin glucoside, peridinin, and in xanthophyll cycle: zeaxanthin ↔ antheraxanthin ↔ violaxanthin [64].

Fig. 3.2. Constitution formulas of (C6–C7) s-cis- (a) and all-trans-beta-carotene (b), together with the labeling of the carbon skeleton (center of inversion i) [70].

Physically carotenoids show yellow to red color according to the number of conjugated double bonds. Carotenoids also have anti-oxidant and photosynthetic properties [71-72].

Moreover, their extended conjugated π-systems have attracted attention to use beta- carotene as electrically conducting wires (Fig. 3.3) [71-73].

Fig. 3.3. Thiol-substituted carotenoids are reported to be significantly more conductive than alkyl chains [71 Fig. 32][73].

Biologically beta-carotene is involved in the process of vision, as biosynthetic precursor of Vitamin A1 or retinal, and is an accessory pigment in photosynthesis – absorbs light that chlorophyll-a does not absorb [70].

Geometrical properties of beta-carotene for gas phase are already known, both experimentally (x-ray) and theoretically, such as structures, bond distances and bond angles [70 Table 1]. Optimized geometry (at the BPW91/6–31G* level) for gas phase shows that s-cis-beta-carotene is 8.8 kJ/mol more stable than all-trans-beta-carotene [70].

Fig. 3.4. Formulas of beta-carotene model systems used to study electronic effects: (a) s-cis- and (b) all-trans- undecene derivative [70].

The density functional theory (DFT) calculations for biological molecules have indicated that the all-trans electronic model system might be stable as a molecular wire (Fig. 3.4) and possibly derivable from the all-trans-beta-carotene by the STM manipulation

[41,45,70]. In this dissertation we have observed five beta-carotene isomers: all-trans- beta-carotene (trans) (Fig. 3.5A), 15-cis-beta-carotene (cis) (Fig. 3.5B), 12-13,12'-13'-di- 29 cis- beta-carotene (twist) (Fig. 3.5C), 13,14'-15‟-di-cis-beta-carotene (V-shape) (Fig.

3.5D), and 14-15,14'-15',15-15',10-11,10‟-11‟-penta-cis-beta-carotene (V-twist) (Fig.

3.5E).

The three all-trans-beta-carotene isomers (Fig. 3.5E) and twelve cis-beta-carotene isomers (Fig. 3.5F) have already been proposed and studied both theoretically and experimentally with other techniques [74-79]. Altogether 272 conformations of beta- carotene have been proposed theoretically [77,79]. While our STM images of trans, cis and twist structures (compare Fig. 3.5A,B,C to F) have been matched with the proposed beta-carotene structures in the literature, the V-shape and V-twist structures discovered in this dissertation work have not yet been proposed nor studied to our knowledge.

C A E

B F

Fig. 3.5. Conformations of beta-carotene that were observed and others: (A) all-trans-beta- carotene [74] (observed, later called trans), (B) 15-cis-beta-carotene [74] (observed, later called cis), (C) 12-13,12'-13'-di-cis- beta-carotene (observed, later called twist), (D) 13,14'-15’-di-cis- beta-carotene (observed, later called V), (E) 14-15,14'-15',15-15',10-11,10’-11’-penta-cis-beta- carotene (observed, later called V-twist), (E) three all-trans-beta-carotene isomers [74], (F) 12 isomers of beta-carotene (mono-cis and di-cis) derived from all-trans-beta-carotene [74].

The relative energy for previous studied isomers (Fig. 3.6A) [74] (three all-trans-beta- carotene isomers (Fig. 3.5E) and twelve cis-beta-carotene isomers (Fig. 3.5F)) indicate that all-trans,s-cis-beta-carotene is the most stable, and “the degree of destabilization of the dicis-beta-carotene isomers may be interpreted as the addition of the individual destabilization degrees of the corresponding monocis isomers” [74], manifesting as approximate additivity in corresponding relative energies (Fig. 3.6B) [74]. 31

Relative Dihedral Isomer Energy Angles dE (kcal/mol) A All-trans-s,s-cis 0 -47.2 47.2 B s-trans,s'-cis 1.42 -169.7 46.2 s-trans 2.75 -169.1 169.1 dE(7,13'-dicis) ~ dE(7-cis) + dE(13-cis) dE(9,13-dicis) ~ dE(9-cis) + dE(13-cis) 7-cis 5.41 -67.6 48 dE(9,13'-dicis) ~ dE(9-cis) + dE(13-cis) 9-cis 1.19 -45.9 46 dE(9,15-dicis) ~ dE(9-cis) + dE(15-cis) 11-cis 5.46 -47.4 47.1 dE(11,11'-dicis) ~ dE(11-cis) + dE(11-cis) 13-cis 1.22 -46.8 47.5 dE(13,15-dicis) ~ dE(13-cis) + dE(15-cis) 15-cis 2.69 -46.9 46.9

7,13'-dicis 6.51 -66 46.9 9,13-dicis 2.11 -48 45.6 9,15-dicis 3.53 -47.8 48.3 9,13'-dicis 2.08 -47.7 48.3 11,11'-dicis 10.66 -46.6 46.6 13,15-dicis 3.67 -47 47.5 Fig. 3.6. Relative energies of beta-carotene isomers and dihedral angles [74].

The percentage composition of 12 known cis isomers [79] derived from all-trans-beta- carotene after artificial isomerization was previously studied [77]. Thermal isomerization for 15 min at 190-200° produced 26.7% all-trans, 46.4% mono-cis and 19.3% di-cis. In the case of photoisomerization, it was found that 47% of the molecules remains as all- trans while 40% mono-cis, and 9.8% di-cis have been transformed [79 Table I].

For the natural structural composition of beta-carotene, the percentage composition of isomers in carotene mixtures from organic palm oil has previously been studied [78]. It 32 was found that 61% of the molecules were trans-beta-carotene while 30.6% were mono- cis , 8.3% di-cis and presence of tri-cis, respectively.

3.2. Chlorophyll-a

Chlorophyll-a (C55H72MgN4O5) has a chemical structure consisting of porphyrin head group and phytyl tail (Fig. 3.7) [2,80]. Chlorophyll-a mainly occurs in photosynthetic complexes in plant leaves (like spinach [81-82]), algae [83], and bacteria [2-5,7,80], and has an absorption spectrum “for all the wavelengths of the visible light except the ones associated with the green light” [2]. The epitaxial growth of Chlorophyll-a on Au(111) shows self-assembly with head-to-head and tail-to-tail alignment [2,80].

The analysis of the crystal structure (X-ray synchrotron diffraction 3.2 Å) of the oxygenic

PS2 core complexes (from cyanobacterium Thermosynechococcus elongatus) suggested a model for the electron transfer between Chlorophyll-a, in chains and antenna, and all- trans-beta-carotene connections [84]. Further study of PS2 core complex composition suggested 36 chlorophyll-a and 9 ± 1 beta-carotene per active centre [85]. Following analysis of re-dissolved crystals of dimeric PS2 core complex showed 0.25 beta-carotene per chlorophyll-a (and 17 ± 1 chlorophyll-a per pheophytin-a), and the corresponding crystal structure showed chlorophyll-a arranged in two layers (close to the cytoplasmic and lumenal sides of the thylakoid membrane) each containing 9 chlorophyll-a [86].

Fig. 3.7. Chemical structure of chlorophyll-a [2].

3.3. Au(111) Substrate

For the experiments described in this dissertation, a Au(111) single crystal surface was used as a template substrate. The substrate sample was cleaned by repeated cycles of sputtering using Ne ions and annealing to 700 K [46-47,50]. The cleanliness of the sample was then checked by STM imaging at 77 K before the molecular deposition.

3.4. Molecular Deposition

The molecules of interest can be deposited on the surface using a home-built Knudson cell (Fig. 3.8) for studies from single molecules to Langmuir-Blodgett (LB) films [12].

Here, the molecules, beta-carotene (trans-beta-Carotene, Type I, Synthetic ~95%, Sigma-

Aldrich Stock # C9750-5G [87]) or chlorophyll-a (Chlorophyll-alpha, Type A from

Spinach, Sigma-Aldrich Stock # C9753-5MG), were placed in a Ta capsule and then heated up to ~450 K via a resistive heater. A thermo couple was attached to the capsule to monitor the source temperature. The cleaned Au(111) sample was directly mounted on 34 top of a button heater attached in a sample holder. The sample holder was placed in a

UHV manipulator with x,y,z motion and rotation facilities. During the molecule deposition process, the sample temperature was held at ~120K to room temperature. The source was positioned approximately 20 cm away from the sample and the molecular depositions were performed in UHV condition.

Fig. 3.8. Schematic of used home built Knudson cell [12]. 35

CHAPTER IV: SINGLE MOLECULE STUDIES

In this chapter we discuss the results of our investigations of single molecules only, specifically beta-carotene molecules studied by UHV-LT-STM on Au(111) surface.

Carotene Conformations

Fig. 4.0. Five mechanically stable conformations of beta-carotene determined in this study, include (left to right and down): trans, cis, twist, v-twist, and v-shape. 36

4.1. Stable Beta-Carotene Conformations on Au(111)

In this section we present five distinct conformations of beta-carotene on Au(111) surface

(Fig. 4.0). To our knowledge, these are the first single molecule images of beta-carotene ever recorded. Moreover, two of the five structures have not been predicted in the literature, and therefore, this is the first discovery of their existence. The presented conformations observed in the STM images did not change most of the time. Thus we classify these conformations as stable during regular scanning mode. As discussed in the next section 4.2, further manipulations were performed to examine these conformations and showed that some conformations survived those manipulations more than others indicating that they were more stable.

4.1.1 Trans Conformation

Fig. 4.1a shows STM topography image of a molecule conformation, which we associate to all-trans-beta-carotene [74], hereafter called trans, chemical structure as discussed in

Chapter 3.1 (Fig. 3.5a). The molecule appears in STM image as two protrusions at both ends corresponding to the two end pi-rings connected by a rod-like feature originated from the central carbon chain of beta-carotene. Moreover, the two end protrusions are positioned opposite site of the central carbon chain. The 3D model (Fig. 4.1b), geometry optimized using Quantum Mechanical system with Parametric Method 3 (PM3) (NDDO -

Neglect of Diatomic Differential Overlap) Hamiltonian via ArgusLab [88-95], shows that the molecular backbone is pretty much flat and in plane, except the last bonds connecting to the end pi-rings, which are also tilted. The ground-state electron density and 37 electrostatic potential mapped surfaces were computed using Quantum Mechanical system with Austin Model 1 (AM1) (NDDO) Hamiltonian [88-92,95] based on the previously mentioned computed optimized geometry and shown in Fig. 4.1c. The PM3 and AM1 computational methods were chosen due to our group familiarity with them in related chlorophyll-a studies [2,80] and their success in organic systems with small errors compared to experiment [88,94,95]. Due to the hexagonal symmetry, Au(111) surface has a hexagonal structure of atomic arrangement. The measurements of the long- molecular axis direction with respect to the surface close-packed directions, i.e. [110] surface directions, reveal the molecular adsorption site. The measurements reveal that the molecule backbone is positioned along a direction 14° rotated from the surface close- packed [110] row (Fig. 4.1d).

A B C D

Fig. 4.1. (a) STM topography images (V = - 1.7 V, I = 0.39 nA) of conformation associated to all- trans-beta-carotene, chemical structure as in Chapter 3.1 (Fig. 3.5a); (b) 3D geometrical model optimized using ArgusLab QM/PM3/NDDO; (c) Ground state electrostatic potential and electron density mapped surfaces calculated using ArgusLab QM/AM1/NDDO based on optimized geometry model in (b); (d) adsorption site model on Au(111) lattice showing that the molecule backbone is 14° from close-packed [110] row (software [96-97]). 38

4.1.2 Cis Conformation

Fig. 4.2a shows an STM topography image of a molecular conformation, where the long axis of the molecule appears bent. In addition, another significant structural change here is that the two end protrusions are located at the same side of the central carbon chain.

The appeared structure is consistent with that of 15-cis-beta-carotene [74], hereafter called cis, chemical structure as discussed in Chapter 3.1 (Fig. 3.5b), and with the 3D geometry optimized (PM3) model (Fig. 4.2b) computed similarly to trans (Fig. 4.1b).

The ground-state electron density and electrostatic potential mapped surfaces (Fig. 4.2c) were also computed (AM1) similarly to trans (Fig. 4.1c), and show that the cis conformation is a dipole. The measurements from the STM image of this molecular structure reveal that the carbon-chain of the molecule is bent for 120 degrees. Moreover, on Au(111) surface, the bent carbon chain aligns along surface close-packed directions as shown in the adsorption model in Fig. 4.2d.

A B C D

Fig. 4.2. (a) STM topography image (V = - 1.5 V, I = 0.17 nA) of conformation associated to 15- cis-beta-carotene, chemical structure as in Chapter 3.1 (Fig. 3.5b); (b) 3D geometrical model optimized using ArgusLab QM/PM3/NDDO; (c) Ground state electrostatic potential and electron density mapped surfaces calculated using ArgusLab QM/AM1/NDDO based on optimized 39 geometry model in (b); (d) molecule model on Au(111) lattice model showing directions of close packed row and molecule backbone (software [96-97]).

4.1.3 Twist Conformation

Fig. 4.3a shows STM topography image of the third molecular conformation. Here the carbon chain between the end protrusions appears with a double tight turns with 120 degree angles. This structure is consistent with that of 12-13,12'-13'-di-cis-beta-carotene, hereafter called twist, chemical structure shown in Fig. 4.3b and also in Chapter 3.1 (Fig.

3.5c), and with the 3D geometry optimized (PM3) model (Fig. 4.3d) computed similarly to trans and cis. The ground-state electron density and electrostatic potential mapped surfaces (Fig. 4.3e), also computed (AM1) similarly to trans and cis, show that the twist conformation is also a dipole like cis. The appearance of a larger protrusion area at one end than the other might be due to different rotational angle of the end pi-ring. A drawback of STM is that one cannot measure the exact tilting angle of the molecule. The adsorption site measurements uncover that the backbone carbon-chain is positioned along surface close-packed row directions. Naturally, the two 120 degree turns of the carbon backbone chain make this adsorption site favorable. A model illustrating the adsorption of this molecule on Au(111) lattice is shown in Fig. 4.3c.

Fig. 4.3. (a) STM topography image of conformation associated to 12-13,12'-13'-di-cis- beta- carotene (V = -1.5 V, I = 0.15 nA); (b) associated chemical structure, also in Chapter 3.1 (Fig. 3.5c); (c) Au(111) lattice model showing that the molecule adsorption sites are along close- packed rows; (d) 3D geometrical model optimized using ArgusLab QM/PM3/NDDO; (e) Ground state electrostatic potential and electron density mapped surfaces calculated using ArgusLab QM/AM1/NDDO based on optimized geometry model in (d) (software [96-97]).

4.1.4 V-Shape Conformation

The fourth stable structure generally observed in STM images of beta-carotene on

Au(111) surface is presented in Fig. 4.4a. This structure can be labeled as 13,14'-15‟-di- cis-beta-carotene with chemical model shown in Fig. 4.4b (also in Chapter 3.1 Fig 3.4d).

Here the backbone carbon chain of the molecule is tightly bent and appears as a „V‟ shape and thus hereafter we will title this structure as „V‟. This structure has never been reported in the literature before. The 3D geometry optimized model, computed as before

(PM3), is shown in Fig. 4.4d. The ground-state electron density and electrostatic 41 potential mapped surfaces shown in Fig. 4.4e, computed as before (AM1), also show that the V-Shape conformation is a dipole like cis and twist. The adsorption site model for

Au(111) lattice shows that the molecule backbone aligns along [211] directions of

Au(111) surface (30°from close-packed [110] rows) (Fig. 4.4c).

Fig. 4.4. (a) STM topography image of conformation associated to 12-13,12'-13'-di-cis- beta- carotene (V = -1.5 V, I = 0.24 nA); (b) associated chemical structure, and also in Chapter 3.1 (Fig. 3.5d); (c) Au(111) lattice model showing that the molecule adsorption sites are along [211] directions (30°from close-packed [110] rows); (d) 3D geometrical model optimized using ArgusLab QM/PM3/NDDO; (e) Ground state electrostatic potential and electron density mapped surfaces calculated using ArgusLab QM/AM1/NDDO based on optimized geometry model in (d) (software [96-97]).

4.1.5 V-Twist Conformation

The last stable structure of beta-carotene on Au(111) found in our study is illustrated in

Fig. 4.5a. Here, the molecule appears as „V‟ shape with an added twist feature in the backbone carbon chain. The structure is labeled as 14-15,14'-15',15-15',10-11,10‟-11‟- penta-cis-beta-carotene, hereafter called V-twist, chemical structure. A possible model is illustrated in Fig. 4.5b (also in Chapter 3.1 Fig. 3.5e). Here, the carbon chain is closely folded at the center resulting in a pi-ring-like shape and producing a protrusion at the center, which gives an impression of a twist. The protrusion at the center can be clearly seen in the 3D geometry optimized model, computed as before (PM3), is shown in Fig.

4.5d. The ground-state electron density and electrostatic potential mapped surfaces shown in Fig. 4.5e, computed as before (AM1), also clearly show the center protrusion, and again this V-Twist conformation is a dipole like cis, twist and v-shape. The adsorption site model on Au(111) lattice shows that the molecule backbone alignment was measured to be 15° from measured direction of the close-packed [110] row (Fig. 5c).

Fig. 4.5. (a) STM topography image of conformation associated to 14-15,14'-15',15-15',10- 11,10’-11’-penta-cis- beta-carotene (V = -1.5 V, I = 0.17 nA), (b) associated chemical structure, and also in Chapter 3.1 (Fig. 3.5e), (c) adsorption site model on Au(111) lattice showing that the molecule backbone is 15° from the close-packed [110] rows (software [96-97]).

4.2. Mechanical Properties of Single Molecules

Mechanical properties of molecules are increasingly important to investigate due to emergent fields of nanotechnology where single molecule devices are being proposed for future applications. Mechanical stability is also critical to understand from the fundamental science point of view. In this dissertation, we use a variety of STM manipulation procedures to qualitatively test how stable are the observed conformations of beta-carotene on Au(111) surface. These investigations confirm the five structures 44 described in previous session as stable conformations. For manipulation statistics please see to Appendix C.

4.2.1 Trans Conformation

In order to test the stability of the molecular conformation, a trans molecule was manipulated laterally across the Au(111) surface (Fig. 4.6) using the STM LM procedure.

During this process, the tip was initially position above one end part of the molecule, then the tip height was reduced. This increased the tip-molecule interaction force. Next, the tip was moved along the surface. During this, the tip height signal was recorded against time. At the final location, the tip was retracted to its normal imaging height. Finally, the result of this manipulation was confirmed by imaging the same area again. As expected, the molecule was moved to a new location. Moreover, the long molecular axis was rotated from its original position. From this experiment, the following information could be deduced: (1) the molecule remained intact during the manipulation process, and

(2) the structure and conformation of the molecule did not change even though it was moved and rotated. In several of other experiments, the molecule was occasionally broken apart during the manipulation or the conformation of molecule was changed if the structure was not stable (see for example V-Wide conformation in section 5.4). The above mentioned two points indicate that the trans conformation of beta-carotene is rather a stable conformation.

A. Before C. Lattice Model

B. After

Fig. 4.6. Proof of stability for trans conformation via lateral manipulation technique shown in STM topography images before (a) and after (b) lateral manipulation (V = 1.7 V, I = 0.12 nA), with corresponding associated interpretation of motion illustrated in a lattice model (c).

4.2.2 Cis Conformation

The stability test of the cis molecular conformation is illustrated in Fig. 4.7. Here STM topography images (Fig. 4.7A) show rotations of the cis conformation about its corresponding Au(111) lattice adsorption sites (Fig. 4.7B) due to electric field induced excitation as a result of scanning and inelastic electron tunneling excitation. The STM images show that the cis conformation was preserved under these rotations indicating that it was stable under these rotations about lattice sites.

4 1

A 2 3

4 1

2 3

Fig. 4.7. (A) STM topography images (V = - 1.5 V, I = 0.17 nA, 11.1x5.4 nm2) showing rotations of the cis conformation about its (B) Au(111) lattice adsorption sites due to electric field induced excitation, as a result of scanning, and inelastic electron tunneling excitation.

4.2.3 Twist Conformation

The stability of the twist conformation was also investigated using the STM tip lateral manipulation technique as before (Fig. 4.8). The STM topography images before and after the lateral manipulation (Fig. 4.8A,B) indicate that the molecule geometry remained the same and that the molecule rotated 120° about its Au(111) lattice adsorption sites

(Fig. 4.8C,D) and shifted laterally as compared to the reference point (right center object 47 in images). Thus this account illustrates that the twist conformation is a stable conformation.

A C

B D

Fig. 4.8. (A-B) STM topography images (V = - 1.5 V, I = 0.15 nA, 8.7 x 3.7 nm2) before and after the lateral manipulation of the twist conformation, and (C-D) its corresponding Au(111) lattice adsorption site models.

4.2.4 V-Shape Conformation

Fig. 4.9 shows the STM topography images (Fig. 4.9A-B) of rotation of the V-shape conformation about its Au(111) lattice adsorption sites (Fig. 4.9C-D) due to electric field induced excitation as a result of scanning and excitation during lateral manipulation.

These STM images show that the V-shape conformation was preserved indicating that it was stable during rotation about lattice sites.

C A

Fig. 4.9. (A-B) Before and after STM topography images (V = - 1.5 V, I = 0.24 nA, 21.7 x 104 nm2) showing rotation of the V-shape conformation about (C) its corresponding Au(111) lattice adsorption sites (solid line – model before, and dotted for after).

4.2.5 V-Twist Conformation

Finally, the V-twist conformation was also investigated using STM lateral manipulation technique (Fig. 4.10). The STM topography images for two lateral manipulations shown in Fig. 4.10A-C indicate that molecule geometry remained the same and that the molecule only rotated 113°counterclockwise about the adsorption sites and shifted laterally in the direction of manipulation (see lattice model in Fig. 4.10D and rotation analysis in Fig. 4.11). This account thus illustrates that this V-twist conformation is also stable for rotations about lattice sites and under the lateral manipulation technique.

D 49

Fig. 4.10. (A-C) S TM topography images (V = -1.5 V, I = 0.17 nA, Lx = 14.25 nm) for two lateral manipulations (V = 80 mV, R = 16 MΩ, I = 5 nA) of the V-Twist conformation, and (D) corresponding proposed lattice models.

A B

Fig. 4.11. Explanation of lateral manipulation signal corresponding to the rotation of the V-Twist conformation (STM images before and after manipulation are shown (Scan: V = -1.5 V, I = 0.17 nA, Lx = 14.25 nm; Manipulation: V = 80 mV, R = 16 MΩ, I = 5 nA)). (A) Molecule rotates 113° counter clockwise direction due to initial excitation of the tip. (B)The tip-height tracing between the two lobes shown in the image along the path 1-2. (C) The molecule hops away from the tip upward so that a sudden drop of tip-height occurs.

CHAPTER V: SINGLE MOLECULE CONFORMATION SWITCHING

Molecular conformation is vital for various functions of carotenoids in their environment and therefore a detailed study of single molecule conformation switching process can elucidate how these molecules transform from one conformation to another. Naturally, the changes of molecular conformations mostly occur by means of thermal excitation or photo excitation processes [77-78]. Molecule conformation could also be switched by means of charge and energy transfer. In this part of the dissertation, we induce various single molecule conformations of beta-carotene on Au(111) surface by using a variety of

STM tip-induced manipulation procedures. The following sub-sections describe the details of observed switching from one conformation to another. For manipulation statistics please see to Appendix C.

5.1. Cis-Trans Isomerization using Tunneling Electrons

The transformation of cis to trans conformation of beta-carotene involves changing a

C=C double bond. Thus, it cannot be realized just by rotation of C-C single bond as in the case of chlorophyll-a conformation switching induced by STM tip on Au(111) [80].

Recently it has been reported that STM tip can be used for isomerization process of single chloronitrobenzene molecules adsorbed on a Cu(111) surface [98]. We have successfully applied an STM tip induced isomerization process to switch a cis to trans conformation of a single beta-carotene molecule for the first time. During this process, the STM tip was positioned above the central location of backbone carbon chain of a cis molecule (Fig. 5.1A), and then the STM feed-back loop was terminated. Thereby the tip 52

is positioned above this location for a certain period with a fixed height and electrons

were injected. After this process, another STM image was taken to check the result. The

STM image illustrated in Fig. 5.1B reveals that the molecule‟s backbone carbon chain

became straight. Thus, we have transformed the previous cis conformation to a trans

conformation. A B

Fig. 5.1. STM topography images (V = -1.7 V, I = 0.17 nA) of the cis conformation (A) switched to the trans conformation (B) under Inelastic Tunneling Electron Excitation procedure performed C with tip positionedCurrent in the center (nA) of the cisvs conformation Time (sec) backbone. 40.00 30.00 20.00 5.2. Cis-Trans Isomerization using Force 10.00 The cis0.00-trans isomerization of beta-carotene can also be realized by using tip-molecule -10.00 0 1 2 3 4 5 6 7 interaction-20.00 force. To date, the isomerization of single molecules induced with an STM -30.00 tip has-40.00 been demonstrated by using tunneling electron induced excitation. Here, we -50.00 demonstrate-60.00 that it could also be done by using force between the STM tip and molecule.

A sequence of STM images in Fig. 5.2 shows that a cis conformation of beta-carotene

molecule (Fig. 5.2A) has been laterally manipulated to form a trans conformation (Fig. 53

5.2B). Here, the STM tip was initially positioned at the center location of backbone carbon chain, and then it was lowered towards the molecule in order to increase the tip- molecule interaction. Then the tip was moved across the surface. During this process, the molecule was transformed to a trans conformation and relocated to a new surface location. Here, the trans conformation was assigned to the new structure because the backbone carbon chain becomes straight. However, only one end pi-ring appeared as a protrusion after isomerization. The other end pi-ring did not appear as a protrusion probably due to a tilted configuration. This will be explained in the following manipulation session 5.4.

A B

V = 70 mV, R = 12 MΩ, I = 6.0 nA C

Tip Height (arb.) (arb.) Height Tip -2 0 2 4 6 8 10 12 14 16 18 20 22 Lateral Distance (nm) Fig. 5.2. Conformation switching from cis to trans using lateral manipulation technique. (a) STM topography images before (V = 1.7 V, I = 0.12 nA, size = 28.5 x 28.5 nm2) and (b) after, with lateral manipulation signal (V = 70 mV, R = 12 MΩ, I = 6.0 nA) (c) showing mostly showing pulling mode. 54

5.3. V-Twist Conformation Switching

In addition to the five stable structures of beta-carotene discussed in section 4.1, beta- carotene can also form less stable structures on Au(111) surface. A lesser stable conformation of beta-carotene is shown in Fig. 5.3a. Here, the molecule roughly appears as a „V‟ shape but with a wide angle, hereafter called V-Wide. Here, we employed STM-

LM procedure to move the molecule across the surface. During this process, the molecule switches to a more stable V-Twist conformation (Fig. 5.3b,h). The lateral manipulation signal (Fig. 5.3c) shows a combination of pushing and pulling lateral manipulation modes [41-43,45-49]. The V-Wide structure observed in STM topography image (Fig. 5.3a, bottom molecule) possibly in contact with a cis conformation is assigned as 13,15,14'-15'-tri-cis-beta-carotene (Fig. 5.3g). This structure can be obtained from the cis conformation (15-cis-beta-carotene) (Fig. 5.3e) by two rotations about the

C=C double bond 13-14 and a single bond of 14‟-15‟. It could also be obtained from the trans conformation (all-trans-beta-carotene) (Fig. 5.3d) by switching first to known

13,15-di-cis-beta-carotene (Fig. 5.3f) conformation [74-77] by two rotations about C=C double bonds of 15=15‟ and 13=14, and then finally switching to the V-Wide structure

(Fig. 5.3g) by one rotation about single bond 14‟-15‟ (Fig. 5.3d-g).

A B

V = 80 mV, R = 16 MΩ, I = 5.0 nA C

-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Tip Height (arb.) (arb.) Height Tip D E Lateral Distance (nm) G H

Fig. 5.3. Switching to V-Twist conformation from V-Wide conformation. (a-b) STM topography images (V = - 1.5 V, I = 0.17 nA) before (a) and after (b) the lateral manipulation (c) (V = 80 mV, R = 16 MΩ, I = 5.0 nA). Chemical structures for V-Wide (g) switching to the V-Twist conformation (h). Possible routes to obtain V-Wide (g) from Trans conformation (d) via cis conformation (e) or via known 13,15-cis-beta-carotene conformation (f).

5.4. Conformation Switching of End Pi-Ring

During our study, some images of beta-carotene show only one protrusion at the end of backbone carbon chain. A protrusion at each end of the backbone carbon chain originated from pi-rings is expected if the molecule has its full chemical structure. In order to confirm that the molecule can appear without end protrusion having its full chemical structure, we perform a set of STM manipulations. A sequence of STM images

(Fig. 5.4a-e) and the corresponding manipulation signals (Fig. 5.4f-i) were obtained 56 through inelastic electron tunnelling (IET) excitation technique [25,45,49-58]. During each manipulation technique the tip was specified to be positioned above the backbone of the molecule as just observed in STM topography image, feedback was turned off, a voltage was applied, and the tunnelling current was recorded. After the manipulation process the feedback was restored and new STM topography image was obtained. The signal in Fig. 5.4f corresponds to a manipulation process after the STM topography image of Fig. 5.4a was taken and the resulting STM topography image of this manipulation was illustrated in Fig. 5.4b. Likewise for the next steps, the signal shown in

Fig. 5.4g corresponds to the manipulation process done on the molecule shown in Fig.

5.4b, and so on. All of these manipulation signals show current step jump behaviour known to correspond to switching phenomena [80,98]. The important information from this manipulation sequence is that the end pi-ring of the molecule can appear either as a protrusion or without a protrusion. For instance, after manipulation from A to B, the end ring appears as a protrusion. By manipulating the molecule in C to D, the end protrusion disappears again. This suggests that the appearance and disappearance of the end protrusion might be due to the tilting of the pi-ring, especially since it is known that different beta-carotene conformations might have different end pi-ring tilt angles relative to the backbone plane (Fig. 3.6A-B) [74]. For instance, the molecule might appear brighter when the pi-ring is lying flat on the surface. In this case, the increased pi- interaction with the substrate can enhance the tunnelling current intensity due to a resonance tunnelling. On the other hand, the tilted pi-ring away from the surface reduces the pi-interaction with the substrate, and decreases tunnelling current intensity. Since the 57

STM images show current density as a measure of apparent height, even though the tilted pi-ring is now physically closer to the tip it would reduce the current intensity, and hence it decreases the apparent height. Or inversely, the tilted pi-ring geometry might provide a higher current intensity due its location closer to the tip. Both situations are possible and conformation of one process or another would require a precise electronic structure calculation including molecule and surface, for example using Density Functional Theory

(DFT) techniques.

A B C D E

F G H I Current vs Time Current vs Time Current vs Time Current vs Time

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

Fig. 5.4. Switching of end pi-ring of V-Wide conformation. (a-e) STM topography images (V = 1 V, I = 0.27 nA) obtained before and after each manipulation (f-i) as indicated by arrows.

5.5. Single Molecule Dynamics

In this section we discuss the dynamics of motion of observed single molecules during lateral manipulation procedure (Fig. 5.5) [33,41-49]. Fig. 5.5 shows how two single molecules (Fig. 5.5a-c) have been pulled apart by moving one away from the other using 58 lateral manipulation technique. The recorded lateral manipulation signal (Fig. 5.5d) shows that in the beginning the tip traced out the molecule ring profile to maintain constant current mode, first moving up slope and then down slope. When the molecule hops to the lattice sites closer towards the tip causing it to quickly retract to maintain constant current mode, which appears in the manipulation signal as a steep increase in tip height at ~ 2.5 nm. Then the tip continued to move laterally once again tracing the down slope of the molecule ring (~2.5 to 3.75 nm), followed by a lateral movement of the tip with a constant height above the surface (~3.75 to 4 nm) until a quick tip retraction occurs at ~ 4 nm. This retraction of the tip indicates that the molecule hops toward the tip in pulling mode [45-46,48].

B D V = 80 mV, R = 16 MΩ, I = 5.0 nA

0 5 10 Tip Height (arb.) (arb.) Height Tip C Lateral Distance (nm)

Fig. 5.5. Single molecule dynamics of two molecules pulled apart. (a-c) STM topography images (V = -1.5, I = 0.15 nA) before lateral manipulation (a) and after (b) (close up in (c)), with recorded lateral manipulation signal (V = 80 mV, R = 16 MΩ, I = 5.0 nA) shown in (d). 59

5.6. Direct Evidence of Beta-Carotene as a Molecular Wire

One of the possible duties of beta-carotene molecules in molecular complexes of photosynthesis systems is that they act as wires [60-61,71-73]. This is further supported by conductivity measurements using break junction techniques and AFM conductivity studies (Fig. 3.3) [71,73,99]. Based on this, beta-carotene molecules have potential applications in nanoscale electronic circuits including as molecular wires for interconnects. But so far, a direct observation of single beta-carotene molecule has never been done before so that it has not been proved directly as a wire. Here, we used an

STM IETS procedure to show that beta-carotene indeed can be act as a wire.

For this experiment, a trans conformation was chosen (Fig. 5.6a) and the inelastic electron tunneling excitation procedure [25,45,49-58] was performed on the lower end pi- ring. Following this manipulation event, an STM topography image was recorded, which showed a change of arrangement in the upper end pi-ring (Fig. 5.6b). Then once again the same excitation procedure was performed on the lower end pi-ring with the following recorded STM topography image showing that the upper end pi-ring completely switched to the right side (Fig. 5.6c). The resulting switch in conformation is symbolized in Fig.

5.6d-f where the initial proposed trans conformation (all-trans-beta-carotene) (Fig. 5.6d)

(STM image Fig. 5.6a) switched during first manipulation to probably 7-cis-beta-carotene intermediate conformation (Fig. 5.6e) (STM image Fig. 5.6b) and then during second manipulation switched to probably 7,8-di-cis-beta-carotene final conformation (Fig. 5.6f)

(STM image Fig. 5.6c). Since the desired switching was initiated in one end pi-ring but 60 actual switching event occurred at the other end of the pi-ring, it is possible only if the injected electrons travel across the backbone carbon chain to excite the other end. This is further supported by the fact that the molecule is weakly bound to the surface as will be explained in the next chapter. Thus we propose that the injected electron in the lower end pi-ring is transmitted through the backbone towards the upper pi-ring, which then triggers the change. This experiment proves that electrons can be easily transmitted through the backbone carbon chain of beta-carotene and thus it will be possible to use it as wires for interconnects.

A B C

trans 7-cis 7,8-di-cis D E F

Fig. 5.6. Proof of beta-carotene as a molecular wire. (a-c) STM topography images (V = - 1.7 V, I = 0.39 nA) before first manipulation (a), after first manipulation (b), and after second manipulation (c). (d-f) The proposed chemical models for resulting switch from proposed trans initial conformation (all-trans-beta-carotene) (d) (STM image in (a)) during first manipulation to probably 7-cis-beta-carotene intermediate conformation (e) (STM image (b)) and then finally during second manipulation switch to probably 7,8-di-cis-beta-carotene final conformation (f) (STM image (c)). 61

CHAPTER VI: MOLECULAR ASSEMBLY

– BETA-CAROTENE CLUSTERS ON AU(111)

In this chapter we study the physical (mechanical) properties and composition of beta- carotene molecular clusters on Au(111) (Fig. 6.0).

Chapter VI Molecular Assembly

A060305.172730.dat V = 1.7 V, I = 0.17 nA

Fig. 6.0. An STM topography image showing overview of Au(111) substrate with beta-carotene molecules appearing isolated and in clusters. The herring-bone reconstruction particular to Au(111) substrate is clearly visible. The top-left to bottom-right smooth step edge makes an angle of 49° with the vertical and was used as a reference for the close packed [110] row.

After deposition of beta-carotene on Au(111), STM images (Fig. 6.0) were first acquired to check the surface condition. Although a few isolated beta-carotene molecules were observed, the surface was mostly decorated with molecular clusters preferentially located at the corners of the surface herringbone reconstruction. This indicates a higher mobility and tendency of cluster formation of beta-carotene on this surface. Analysis on the structure of isolated single beta-carotene molecules reveals that the molecules are mostly in trans conformation with an exception of a few cis. In this chapter, we attempt to qualitatively determine the attraction between the molecules inside the clusters as well as the interaction of clusters with the substrate. For the former case, extraction of single beta-carotene molecules have been performed while for the latter case, lateral manipulation of the whole clusters have been done. In addition, we provide conformation statistics of beta-carotene inside the clusters on Au(111).

6.1. Single Molecule Extraction

In this section we discuss how the molecules were extracted from molecular clusters (like in Fig. 6.0) using the lateral manipulation technique [33,41-49] by examining the resulting lateral manipulation signals, and also how much strength was required to extract them qualitatively.

STM topography images (Fig. 6.1) show two events of partial pullout of the cis conformation from molecular clusters using the lateral manipulation technique where one molecular lobe (end pi-ring) was pulled out from cluster while the other end was still remained close to the cluster due to attractive interactions between the molecules. The resulting lateral manipulation signals for these two events (Fig. 6.1c,d) showed that as the tip initially approached the molecule it traced out the molecule end pi-ring profile and then continued laterally following the surface without any indication of molecule following at the beginning. This tip movement continued until the tip was at ~ 16.1 Å

(14.2 Å for the second case) when a sudden significant tip retraction was detected

(marked in Fig. 6.1c,d) indicating that the molecule hopped toward the tip due to the tip- molecule attractive interaction, which overcome the surface barrier and the molecule- cluster cohesive force [42-43,45-46,48-49]. The STM topography images (Fig. 6.1a2,b2) acquired after the process showed that one end of the molecule (end pi-ring) was pulled away from the cluster (see in STM images Fig. 6.1a1,a2 and the corresponding manipulation signals in Fig. 6.1c,d), while the other end of the molecule (end pi-ring) remained still close to the cluster almost as before manipulation (compare before and 64 after STM images Fig. 6.1a1-a2,b1-b2). Fig. 6.1e summarizes the lateral manipulation parameters, the distances of major jumps of the specified molecule end pi-ring (as just discussed) and its distances from the cluster (i.e. to the closest lobe in the cluster).

We used the same tunnelling parameters and the same tip in these two interaction events.

If we approximate the lateral forces required to produce the observed major jumps as proportional to the jump distances, i.e.: FL ~ d, then we can estimate the ratio of lateral forces as a ratio of corresponding major jump distances, i.e.: (Eq. 8.1)

FL1 / FL2 = d1 / d2 (Eq. 8.1)

Thus we can estimate how much stronger was the force in one event versus another via the relation (Eq. 8.2):

FL2 = (d 2 / d 1) F L1 (Eq. 8.2)

In this case the lateral force in event A is greater than in B by 13% as follows (Eq.8.3):

F L_A = ( d_A / d_B ) FL_B = ( 16.1 / 14.2 ) / FL_B = 1.13 FL_B (Eq. 8.3)

This value was measured from Fig. 6.1c,d, and e.

The full molecule pullout sequence corresponding to the STM images in Fig. 6.1 is depicted in Fig. 6.2, showing that a cis conformation was pulled out from the edge of the molecular cluster, using the lateral manipulation technique, only after several attempts were made all with the same manipulation parameters.

For first three attempts (Fig. 6.2A-D) were not successful and only on the fourth try (Fig.

6.2D) did one end of the molecule (end pi-ring) pull out (Fig. 6.2E). During this sequence the molecule sprang back to the cluster twice during the lateral manipulations

(Fig. 6.2E-F and 6.2G-H), but was eventually pulled out completely (Fig. 6.2 J-K) with the manipulation signal in Fig. 6.2J showing a regular well known pushing manipulation mode (Fig. 2.3 (2)).

Displayed are the initial STM images before each manipulation with the manipulation direction overlaid (Fig. 6.2A-J left images) and the corresponding manipulation signal shown beside each image (Fig. 6.2A-J right plots). The last STM image in the sequence is shown in Fig. 6.2K. 66

A1 B1

A2 B2 A2 B2

C D C D

V = 70 mV, R = 10 MΩ, I = 7.0 nA V = 70 mV, R = 10 MΩ, I = 7.0 nA V = 70 mV, R = 10 MΩ, I = 7.0 nA V = 70 mV, R = 10 MΩ, I = 7.0 nA E Set Major Jump Distance Distance to Cluster R V I Before Manipulation A ~ 16.1 A ~ 10 A 10 MΩ 70 mV 7.0 nA B ~ 14.2 A ~ 9.6 A 10 MΩ 70 mV 7.0 nA Fig. 6.1. Analysis of partial pullout of cis conformation from molecular cluster. (a1-a2,b1-b2) STM topography images (V = 1.7 V, I = 0.12 nm) of two partial pullout sequences with start and direction of lateral manipulation (a1, b1) and images after manipulations (a2, b2), with corresponding lateral manipulation signals (c) and (d) (V = 70 mV, R = 10 MΩ, I = 7.0 nA) showing major jump of molecule’s one end pi-ring toward the tip. (e) Summary of lateral manipulation parameters and distance measurements for two sequences.

Fig. 6.2. Continues on the next page. 68

Fig. 6.2. The full molecule (cis conformation) pullout lateral manipulation sequence corresponding to STM images in Fig. 6.1. All lateral manipulation parameters there the same. 69

6.2. Beta-Carotene Clusters

Here we discuss the statistics of different beta-carotene molecular conformations inside clusters like shown in Fig. 6.2.0 below. Carotene Clusters

Fig. 6.2.0. An STM topography image showing overview of Au(111) substrate with beta-carotene molecules appearing in clusters. The herring-bone reconstruction particular to Au(111) substrate is clearly visible.

Using the proposed five conformation of beta-carotene we have analyzed the composition of molecular clusters to determine how many beta-carotene molecules of each type were present (Fig. 6.3 and Fig. 6.4). An example of analysis of typical clusters (Fig. 6.3) shows the chemical structures of the proposed conformations overlaid onto the recorded

STM topography images of two clusters (Fig. 6.3a1,b1), where 3D view and 70 approximation of molecular volume (charge density) were used to ease matching of conformations within the clusters (Fig. 6.3a2,b2). The cluster statistics (Fig. 6.4) show that out of 11 clusters analyzed the distribution of beta-carotene proposed conformations within the clusters was 14.5±2.6% Trans, 34.2±6.3% Cis, 9.2±1.7% Twist, 14.5±2.6% V-

Shape, 27.6±5% V-Twist, and 18.3% Unknown (total error), with error bars are based on this total error (for details see Appendix A).

A2 B2

Fig. 6.3. Composition analysis of two typical clusters showing the chemical structures of the proposed conformations overlaid onto the recorded STM topography images of two clusters (a1,b1), where 3D view and approximation of molecular volume (charge density) were used to ease matching of conformations within the clusters. 71

Fig. 6.4. Cluster composition statistics (error bars are based on total error, for details see Appendix A).

The comparison of our observations of beta-carotene cluster percentage composition with literature is summarized in Table I and Fig. 6.5. The tabulated composition percentages reported in literature have already been introduced in Chapter III section 3.1, and include natural oil composition [78], artificial photoisomerization [79], artificial thermal isomerization [79], and calculated relative energies of isomers [74]. The table shows that the concentration of trans-beta-carotene decreases from 61% in natural oil, to 47% in photo-isomerization, to 26.7% thermal isomerization, and to 14.5±2.6% our on Au(111) study, while opposite increasing trend is observed for other isomers, since more trans isomerizes, as shown in Fig. 6.5. The concentration of cis (mono-cis) isomers goes though a maximum, that is increases from 30.6% in natural oil, to 40% in photoisomerization, and to 46.4% in thermal isomerization, but then decreases to 34.2±6.3% 72 our value on Au(111), which can be explained as more mono-cis are isomerizing to other isomers with the relative energy of the new isomer approximately given by the addition of the relative energies of the related mono-cis isomers that are the necessary transition points along the isomerization path (e.g. dE(7,13‟-di-cis) ~dE(7-cis) +dE(13-cis))

(Chapter III section 3.1 Fig. 3.6 [74]). The increase in non-all-trans isomers can be explained using energy supplied to the molecules and relative isomer energies, that is, the higher is the sufficient energy and the longer it is available the more probable that the trans molecules will isomerize since all-trans has the lowest energy compared to other isomers, as can be seen the Table I Relative Energies (also Chapter III section 3.1 Fig. 3.6

[74]). Thus, the tabulated results from experiments indicate that in natural oils the energy supplied is the lowest, followed by photo-isomerization – low energy supplied, then thermal isomerization – medium energy supplied, and in our study on Au(111) having the highest energy supplied. The reason for our study can be explained as a result of the combinations of initial thermal heating during deposition and energy transfer between molecules during agglomeration into clusters due to prevalent dipole nature of beta- carotene non-trans isomers as indicated for cis, twist, v-shape and v-twist in Chapter IV sections 4.1.2-4.1.5. The concentration dynamics for different temperatures can understood by converting the tabulated relative energies for each isomer given all-trans ground state into isomerization probabilities, which is the expected concentration percentage, using Statistical Mechanics Probability = exp(-dEi/kbT) / Sum_i(exp(- dEi/kbT)) (Callen, H, eq.16.11 page 351) [100]. The time dependence should also be considered, since longer times with sufficient energy will result in more isomerizations. 73

Thus, finally, to understand the global dynamics of isomer concentrations one should

employ the temperature and time dependent probabilistic analysis.

Table I: The comparison of our observations of cluster percentage composition with literature. Isomer Our results all-trans- all-trans-beta- Natural Calculated relative Type of beta- beta- carotene artificial composition energies of beta-carotene carotene carotene photoisomerization of beta- isomers [74]. on Au(111) artificial in solution [79 carotene thermal Table I] from Can be converted to isomerization organic Percentage using Boltzman for 15 min at palm oil Statistics: 190-200° in [78] Probability= exp(-dEi/kbT) solution [79 /Sum_i(exp(-dEi/kbT)) Table I] (Callen.eq16.11.p351)[100] Energy Highest Medium Low Lowest Supplied trans 14.5±2.6% 26.7% 47% 61% 0 All-trans-s,s-cis Trans all-trans all-trans trans 1.42 s-trans,s'-cis 2.75 s-trans mono-cis 34.2±6.3% 46.4% 40% 30.6% 5.41 7-cis Cis mono-cis mono-cis mono-cis 1.19 9-cis (mono-cis) 5.46 11-cis 1.22 13-cis 2.69 15-cis di-cis 23.7±4.3% 19.3% 9.8% 8.3% 6.51 7,13'-dicis (total di-cis: di-cis di-cis di-cis 2.11 9,13-dicis 9.2±1.7% Twist + 3.53 9,15-dicis 14.5±2.6% 2.08 9,13'-dicis V-Shape) 10.7 11,11'-dicis 3.67 13,15-dicis other-cis 27.6±5% presence of V-Twist tri-cis (penta-cis)

Fig. 6.5. Percent composition trends (vertical axis) for beta-carotene isomers for increasing supplied energy (horizontal axis): (1) natural oil [78], (2) photo-isomerization [79], (3) thermal isomerisation [79], (4) our study of beta-carotene on Au(111) (thermal, dipole).

6.3. Cluster Manipulation

In this section we apply lateral manipulation technique [33,41-49] on the entire molecular cluster instead of single molecule. Fig. 6.6 shows STM topography image (Fig. 6.6a) of molecular cluster with a straight line showing the orientation of the upper cluster. The

STM lateral manipulation technique (signal Fig. 6.6c) was performed in the direction and distance specified by the arrow. The resulting STM topography image (Fig. 6.6b) shows that the entire upper cluster was shifted to the left pivoting at the top of the cluster shown by a straight line now oriented in the new direction of the cluster orientation and an arrow indicating pivoting. The lateral manipulation signal (Fig. 6.6c) shows a combination of pushing and pulling modes [42-43,45-46,48-49]. The qualitative conclusion of this 75 observation is that the entire cluster was shifted mostly intact (preserving its structural integrity), even across herringbone reconstruction. This experiment indicates that the molecule-molecule interactions within the cluster are stronger than the molecule- substrate binding, and that the tip-cluster interactions are sufficiently strong enough to provide lateral forces that overcome lattice hoping barriers, but are sufficiently weak not to rip the cluster apart.

A B

Fig. 6.6. Cluster manipulation using Lateral Manipulation technique. (a-b) STM topography images (V = -1.5 V, I = 0.15 nA) of molecular cluster before (a) and after (b) the lateral manipulation, signal in (c), with straight lines in STM images indicating the orientation of molecular cluster before and after, and arrows indicating the direction and distances of lateral manipulation in (a) and pivoting in (b). 76

CHAPTER VII: BETA-CAROTENE – CHLOROPHYLL-A COMPLEXES

7.1. Previous STM Experiments

To date, there are no reports on STM studies of chlorophyll-a and beta-carotene mixture in the literature, even though STM studies on just chlorophyll adsorbed on Au(111) have been reported. The controlled conformation switching of chlorophyll-a on Au(111) surface using the STM tip have been performed and it showed that the chlorophyll-a molecule can be switched between four surface conformations by injecting a single electron into specific bonds using IETS [80]. More experiments have studied chlorophyll-a film using STM [101-103] and have showed well-ordered structures [101].

The STM experiments of bacteriochlorophyll-c [104-106], including on HOPG [104-105] also showed molecular self-assembly. Another STM experiment in UHV of chlorophyll on the mesoporous nanocrystalline TiO2 films (for dye-sensitised photovoltaic cells) showed a film band gap of 3.2 eV [107-108].

7.2. Our Results

In this section we report on our studies of beta-carotene mix together with chlorophyll-a as observed on Au(111) surface using LT-UHV-STM (Fig. 2.1 [30]) conducted at liquid nitrogen temperature of ~77 K.

For this experiment, we first deposited chlorophyll-a (Chlorophyll-alpha, Type A from

Spinach, Sigma-Aldrich Stock # C9753-5MG) on Au(111) surface using similar 77 technique as for beta-carotene previously mentioned in Chapter 2 (deposited on clean sample at 450 K, sample cleaned with repeating 4 cycles of sputtering and annealing, source first outgassed till 373 K and turned off right away about 1 hour before). The resulting STM topography images at ~77 K (liquid nitrogen) confirmed the chlorophyll-a structure and self-assembly and less than one Langmuir-Blodgett layer deposition. Here both molecular clusters and empty surface terraces were observed.

Next, we additionally deposited beta-carotene on the chlorophyll-a/Au(111) sample at

400 K using similar technique for beta-carotene previously mentioned in Chapter 2.

The resulting STM topography images of the mixture showed three distinct regions: chlorophyll-a well-ordered self-assembly, highly mobile beta-carotene regions (high intensity), and chlorophyll-a + beta-carotene mixture (Fig. 7.1 left). The close-up STM topography image of the mixed region showed recognizable single molecules of chlorophyll-a with the same structure as in the adjacent well-ordered self-assembled chlorophyll-a region (Fig. 7.1 right). The herring-bone surface reconstruction particular to Au(111) surface was also visible through the mixture (Fig. 7.1 left).

Fig. 7.1. (Top) Large scale STM topography image of the chlorophyll-a and beta-carotene mixture (77 K, 1.2 V, 0.21 nA) showing three distinct regions: chlorophyll-a self-assembly, highly mobile beta-carotene regions (high intensity) and chlorophyll-a + beta-carotene mixture. (Bottom) A close-up STM topography image (1.2 V, 0.12 nA) of the left image showing recognizable single molecules of chlorophyll-a in the chlorophyll-a + beta-carotene region with the same structure as in the adjacent self-assembled chlorophyll-a region. 79

Further content composition analysis of beta-carotene + chlorophyll-a mixture regions was performed by visually inspecting the mixture regions of STM topography images for different conformations of single beta-carotene molecules (as identified in this dissertation: trans, cis, twist, V-shape and V-twist) and single chlorophyll-a molecules

(from previous study: straight, bent 1, bent 2, bent 3 [80]). The resulting summary statistics across 9 mixtures (Fig. 7.3-5) show that 56.4±10.1% of examined mixtures was chlorophyll-a (42.7±7.7% (75.6±13.6% out of chlorophyll-a only) as straight conformation, 4.7±0.9% (8.4±1.5%) as bent conformation 1, 3.2±0.6% (5.6±1.0%) as bent conformation 2, 5.9±1.1% (10.4±1.9%) as bent conformation 3 (Fig. 7.4)) and

43.6±7.8% as beta-carotene (24.4±4.4% (56.0±10.1% out of beta-carotene only) as

Trans, 6.8±1.2% (15.5±2.8%) Cis, 1.8±0.3% (4.1±0.7%) Twist, 6.8±1.2% (15.5±2.8%)

V-Shape, 3.8±0.7% (8.8±1.6%) V-Twist) (for details see Appendix B).

Fig. 7.2 Left shows one of the analyzed STM topography images, where beta-carotene and chlorophyll-a conformation chemical models have been overlaid onto the image to mark the identified molecules, with approximation of molecular volume (charge density) of overlaid molecules shown in Fig. 7.2 Right. The regions marked A, B and C in Fig.

7.2 Right show chlorophyll-a porphyrins aligned towards beta-carotene beta-ionine rings due to electrostatic attraction between them, and due to electrostatic repulsion between delocalized electrons within beta-carotene backbone and chlorophyll-al phytyl tail.

A B

Fig. 7.2. (Left) STM topography image of mixture region of beta-carotene and chlorophyll-a, beside chlorophyll-a self-assembly, with the content of mixture region analyzed for beta-carotene and chlorophyll-a conformations. (Right) Recognized conformations highlighted with regions A, B and C indicating chlorophyll-al porphyrins aligned towards beta-carotene beta-ionine rings.

Fig. 7.3. Content percent composition of chlorophyll-a and beta-carotene mixture regions (error bars are based on total error, for details see Appendix B). 81

Fig. 7.4. Content percent composition of chlorophyll-a within the mixtures of chlorophyll-a and beta-carotene (error bars are based on total error, for details see Appendix B).

Fig. 7.5. Content percent composition of beta-carotene within the mixtures of chlorophyll-a and beta-carotene (error bars are based on total error, for details see Appendix B). 82

CHAPTER VIII: CONCLUSION

8.1. Beta-Carotene

In summary we report the observation and manipulation of five beta-carotene single molecule isomer conformations on Au(111) substrate studied by Scanning Tunneling

Microscope in Ultra High Vacuum at Low Temperature of 77 K and 4.2 K. To our knowledge these are the first observations of these beta-carotene conformations at a single molecule level. Each conformation was tested for mechanical stability as single molecule using STM lateral manipulation or Induced Electron Tunneling Excitation resulting in translation or rotation of molecules, implying that observed conformations were stable. It was also possible to switch conformation from trans to cis using Induced

Electron Tunneling Excitation technique, where tunneling electrons causing an excitation in the molecular backbone sufficient to overcome the potential barrier of switching to a new cis conformation. We also observed the switching of single trans molecule end group (beta-ionine ring) from one side of the backbone to the other by inducing an excitation in the other end group (beta-ionine ring), which remained unchanged afterwards, using the Induced Electron Tunneling Excitation technique, implying that the excitation travelled from one beta-ionine end group to the other through molecular backbone, with surface dissipation being not sufficient to impede the excitation transfer necessary for switching since the molecules physisorb and the molecule-subrate interaction was observed to be weak compared to inter-molecular interaction within cluster as mention below. This excitation transfer observation implies a potential 83 possibility of using beta-carotene molecules, especially as trans, as molecular wires, especially since the thiol-substituted carotenoid structurally similar to beta-carotene was reported to be significantly more conductive than alkyl chains (Fig. 3.3) [71,73].

Furthermore, the studies of redox reactions during an electron transfer in photosynthesis

Photosystem II have also suggested that beta-carotene acts as a intermediate and a

"molecular wire" as an extended pi-electron-conjugated system [61].

After the deposition only few isolated single molecules have been observed on Au(111), and most molecules instead formed clusters. It was possible to pull out single molecules from such clusters, in some cases after several attempts, for further study, and the corresponding single molecule dynamics were analyzed in detail. The fact that sometimes it took several attempts to pullout the molecules from the clusters with the same parameters implies that frictional (adhesion) effect is present as a molecule-cluster interaction, resembling static coefficient of friction in classical mechanics but on single molecule level. It was also possible to laterally manipulate (displace) part of an entire cluster while mostly preserving its structural integrity. This implies that the intermolecular interaction within clusters is stronger than the molecule-substrate interaction. The clusters were analyzed further for composition of observed isomer conformations and the statistics, which revealed that the originally deposited trans conformation was a small minority and cis and other conformations were highly present, resulting in a rich but disordered variety of conformations within the clusters. This indicates that, in addition to thermal isomerization during deposition, molecules also preferentially rearrange into different conformations within clusters, probably to 84 minimize potential energy by maximizing (optimizing) intermolecular interaction. This observation possibly implies that beta-carotene molecules can aggregate into disordered multi-conformational (multi-isomer) clusters or vesicles in 2D or 3D biological systems where molecule concentration is not sufficient to produce layering.

8.2. Beta-Carotene and Chlorophyll-a

We also report on observation of beta-carotene and chlorophyll-a mixture studied similarly on Au(111) substrate with less than one Langmuir-Blodgett monolayer coverage, again studied using LT-UHV-STM at 77 K. The chlorophyll-a molecules were deposited first and showed cluster self-assembly with less than one Langmuir-Blodgett monolayer coverage as observed in previous study [80]. The following additional deposition of beta-carotene molecules resulted in three identifiably distinct regions: chlorophyll-a well-ordered self-assembly, highly mobile beta-carotene regions (high intensity), and chlorophyll-a + beta-carotene mixture, with the Au(111) herring-bone surface reconstruction still visible through the mixture. It was possible to recognize single chlorophyll-a molecules in the mixed regions with the same structure as in the adjacent self-assembled chlorophyll-a regions. The content of mixtures was further analyzed for different chlorophyll-a conformations as previously reported [80] and previously mentioned single molecule beta-carotene observed conformations, and statistics showed that about half of mixture was chlorophyll-a, with strong majority as straight conformation and other bent conformations occurring in small minorities. The other half was beta-carotene with about half as trans and about fifth as cis and others as small minorities. Inspection of molecular alignment suggests that porphyrin unit of 85 chlorophyll-a tends to align towards beta-carotene beta-ionine rings probably due to electrostatic attraction between them and electrostatic repulsion between delocalized electrons within beta-carotene backbone and chlorophyll-a phytyl tail.

The difference between our observations and biological mixtures, like in photosynthesis

[60,63-68,81-82,84-86,109-111], is that chlorophyll-a to beta-carotene concentration found here is about 50-50% (half-half = 1 : 1) versus 80-20% (chlorophyll-a : beta- carotene = 4 : 1) found in [84-86]. 86

APPENDIX A: CLUSTER COMPOSITION STATISTICS (BETA-CAROTENE)

Fig. A.1 tabulates cluster composition statistics as number and percent of occurrences of

each beta-carotene stable conformation that were found within the examined 11 clusters.

The total error is ~18.3% determined from the number Unknown configurations. The

individual % Error for each confirmation is then calculated by first determining # Error

based on Total Error and then converting that to percentage based on total number of 34.2% 35% known configurations. The plot is shown27.6% Chapter VI Fig. 6.4. In addition to the already 30% Cluster 25% presented examples of cluster analysis, such as examined cluster 1 (Chapter VI Fig. 6.3 20% Composition 14.5% 14.5% 15%B1-B2) and examined cluster 5 (Chapter VI Fig. 6.3 A1-PercentA2), the rest of the examined 9.2% 10% 6.3% 5.0% Statistics 5%clusters 22.6% to 11 used for composition1.7% 2.6% analysis are presented here in Fig. A.2-3.

0% Trans Cis Twist V V-twist

# Total # Error % Total % Error

Trans 11 2 14.5 2.6

Cis 26 4.8 34.2 6.3

Twist 7 1.3 9.2 1.7

V-Shape 11 2 14.5 2.6

V-twist 21 3.8 27.6 5

Unknown 17 18.3 % <-- Total Error

Total All 93

Total Known 76

Fig. A.1. Cluster composition statistics.

C.2 C.3

C.4

C.8 C.7

Fig. A.3. (C.2-C.8) Beta-Carotene clusters 2 to 8 (except cluster 5). C.9 C.10 C.11

Fig. A.4. (C.9-C.11) Beta-Carotene clusters 9 to 11 . 88

APPENDIX B: MIXTURE COMPOSITION STATISTICS

(BETA-CAROTENE + CHLOROPHYLL-A)

Similarly to beta-carotene cluster composition analysis in Appendix A, here the percent composition of selected mixtures of beta-carotene and chlorophyll-a are tabulated in Fig.

B.1 and plotted in Chapter VII Fig. 7.3. The percent composition of individual molecules of beta-carotene and chlorophyll-a separately are also tabulated (Chl/BC column) and plotted in Chapter VII Fig. 7.4-5. In all cases the error bars are calculated based on total error, like in beta-carotene statistics Appendix A. Likewise, in addition to the already presented example of composition analysis of mixture 1 in Chapter VII (Fig. 7.2), the rest of the analyzed mixtures 2 to 9 used for composition statistics are presented here in Fig.

B.2-6 (M.2-9).

Fig. B.1. Composition statistics for mixtures of beta-carotene and chlorophyll-a. 89

M.2

M.3 Fig. B.2. (M.2-M.3) Beta-carotene + chlorophyll-a mixtures 2 and 3.

M.4

M.5 Fig. B.3. (M.4-M.5) Beta-carotene + chlorophyll-a mixtures 4 and 5. 90

M.6

Fig. B.4. (M.6) Beta-carotene + chlorophyll-a mixture 6.

M.7

M.8

Fig. B.5. (M.7-M.8) Beta-carotene + chlorophyll-a mixtures 7 and 8.

Fig. B.6. (M.9) Beta-carotene + chlorophyll-a mixture 9.

APPENDIX C: MANIPULATION STATISTICS

Below Fig. C.1 shows count of occurrences for the presented manipulations in this dissertation with associated figures, such as single molecule rotation/translation manipulation, single molecule switching, single molecule pull-outs from clusters, and cluster manipulation, however, more occurrences have been recorded. Specifically, for reference, the full set of data and manipulation files relevant to this dissertation are counted in Fig. C.2, showing for example that over 4 experiments 424 Lateral

Manipulation (LAT) at 4.2 K (liquid helium temperature) files have been recorded, some analyzed and presented here, while others unsuccessful or not analyzed, or analyzed but not included here.

Fig. C.1. Manipulations presented in this dissertation.

Fig. C.2. Count of STM data files relevant to this dissertation. 94

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