Nilusha Perera Thesis

Study of Far-Side Geometrical Enhancement in Surface-Enhanced Raman Scattering

A thesis submitted for the degree of

Doctor of Philosophy

Nilusha Perera

Centre for Quantum and Optical Science

Faculty of Science, Engineering and Technology

Swinburne University of Technology

Melbourne, Australia

2017

“One of the basic rules of the universe is that nothing is

perfect. Perfection simply doesn't exist... Without

imperfection, neither you nor I would exist.”

Stephen Hawking

Dedication

This thesis is dedicated to my parents (Thaththa Edward and Amma Desy) who have provided me endless love and courage to follow my dream over the years and to my loving husband Rajith who has always been with me throughout this wonderful journey of PhD.

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Abstract

Surface-enhanced Raman scattering (SERS) is a promising method for detection of biomolecules and trace chemicals. The well-established concepts of electromagnetic and chemical enhancements provide the theoretical background of SERS. Over the years since the discovery of SERS, a few research groups have observed an additional enhancement in the SERS signal when exciting the SERS substrate through an underlying transparent dielectric substrate (referred to here as ‘far-side geometrical enhancement’). The experimental evidence was not only limited to planar SERS substrates, but also observed for some SERS-active angled fibre tips.

The first significant attempts to explain the origin of this geometrically-enhanced SERS signal appeared in 2013. The supporting information in those works was limited to a single excitation wavelength and provided only qualitative agreement between the proposed theories and the experimental results. Consequently, there remains a need to more carefully study this extra enhancement by addressing some of the gaps in the proposed theories, and comparing the theoretical predictions against more detailed experiments. This information will be useful for a range of applications where it is necessary to understand the effective optical behaviour of thin plasmonic films, including optical windows and in designing miniaturised optical fibre sensors for remote sensing applications.

Therefore in this thesis, the far-side geometrically-enhanced SERS signal was further explored in a more rigorous theoretical and experimental basis. Nanostructured silver films fabricated on transparent substrates, using oblique angle deposition (OAD) at an angle of 86° with a nominal thickness of 100 nm, were used as the SERS substrate. The effective optical constants of transparent substrates and the nanostructured Ag layer were carefully determined by spectroscopic (standard) ellipsometry and generalised Mueller matrix ellipsometry, respectively. The generalised Mueller matrix ellipsometry data analysis revealed a uniaxial optical response in the Ag OAD86° 100 nm film, with the optical axis inclined towards the source of the deposition vapour flux. The wavelength-dependent effective optical constants as determined by the uniaxial model were represented by complex numbers with real and imaginary parts representing refractive index and extinction coefficient, respectively. The effective optical constants differed substantially

v from those of the bulk silver, with a significant localised surface plasmon resonance (LSPR) peak in the extinction coefficient.

As has been identified previously, this geometrical enhancement appears to follow the E 4 approximation in SERS. Therefore, the wavelength-dependent complex effective optical constants were incorporated in the Fresnel equations to calculate the corresponding electric fields at the metal-dielectric boundary for near-side and far-side excitation of the SERS substrate. The theoretical model derived with the complex reflection and transmission coefficients in the Fresnel equations consistently showed a geometrically-enhanced SERS signal for the far-side excitation compared to the near-side. Ellipsometrically-determined optical constants of Ag OAD86° 100 nm films deposited on glass and sapphire substrates were used to model the substrate material dependence. Further, the extended Fresnel model predicted that the highest geometrical enhancement factors should arise close to the LSPR peak of the sample, with a significant red-shift of the peak for increasing refractive index of the underlying substrate.

Silver OAD86° 100 nm substrates functionalised with thiophenol were used to study the dependence of the geometrically-enhanced SERS signal on the excitation wavelength, supporting substrate material and excitation angle of incidence. The experimental SERS results confirmed that the highest geometrical enhancement factors up to 3.2-3.5 are seen closer to the LSPR of the metallic nanostructures, with a slight red-shift from the theoretical predictions. Both the theoretical predictions and experimental results generally agree with each other for the dependence on the wavelength and substrate material, although precise quantitative comparison was not achievable due to the relatively large variability (± 10-15%) in the SERS measurements. However, increasing angle of incidence showed a decrease in the experimental geometrical enhancement factor, which contradicts the gradual increase predicted by the extended Fresnel model. Further validation of the extended Fresnel model will require substrates with reduced variability and avoiding systematic errors when measuring the dependence on angle of incidence.

In view of the results of this study, it appears likely that the far-side SERS signal is amplified by the net enhancement in the electric field at the metal-dielectric substrate interface compared to the near-side excitation. Given this improved understanding of the far-side geometrical SERS enhancement, the potential for further signal amplification and optimisation for practical sensing applications can now be considered. This study also provides a pathway to use ellipsometry to characterise different SERS substrates when investigating the far-side geometrically-enhanced SERS signal.

Acknowledgements

This document is not only a thesis but also represents the most challenging, yet inspiring and exciting chapter of my life. I have been blessed enough to be surrounded by lovely people and the work of this thesis would not have been possible without the support of most of them.

A big thank you to my principal coordinating supervisor Prof. Paul Stoddart who has not just been my supervisor but also my mentor. I am forever grateful to have met a person like you who gave me the space and time to mature during tough times of the PhD when I was planning to quit. Thank you Paul for guiding me through the challenges and problems of my project with your expertise knowledge and experience. No meeting was ever boring and you always had the power to lift my spirits when the days were dark and gloomy. I could not have hoped for a better supervisor for my PhD project. It has been a privilege to work with a person like you, and your inspirational thoughts will forever be a treasure of my life.

I would like to thank Prof. Russell McLean, Prof. Margaret Reid and Dr. Qiongyi He from Centre for Quantum and Optical Science (CQOS) for introducing me to the Applied Optics Group and giving me the opportunity to work with them.

A special thank you to Prof. Saulius Juodkazis for the advices and support given during my PhD candidature as the coordinating supervisor of the project. It has been a great experience to share the work with the Applied Plasmonic Group at Centre for Micro- Photonics (CMP).

I kindly thank Mr. Keith Gibbs for all the discussions we had, for the time you especially dedicated to read my writing and your efforts to provide me constructive feedback. Some days were hard and it was not easy to convince you with my vocabulary. It has been a pleasure to have met an enthusiastic physicist like you to discuss my work. Thanks again for your insights and suggestions.

I want to say thank you to Prof. Sally McArthur, Prof. Peter Cadusch, A/Prof. Scott Wade, Prof. Simon Moulton and Dr. Michelle Dunn for their constructive feedback during my presentations at group meetings.

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I also owe a special thanks to my panel members of the PhD progress reviews including A/Prof. Scott Wade, Prof. Simon Moulton, A/Prof. James Chon and Prof. Damien Hicks for their feedback and suggestions based on diverse experience and immense knowledge on different research fields.

I am grateful to my Swinburne colleagues Ms. Jennifer Hartley, Dr. Ricardas Buividas, Dr. Gediminas Seniutinas, Dr. Gediminas Gervinskas, Dr. Chiara Paviolo, Dr. Nirali Shah, Dr. Smitha Jose, Dr. Myintzu Hlaing, Dr. Muhammad Awais, Dr. Prince Surendran, Ms. Emma Carland, Dr. Hitesh Pingle, Mr. Nainesh Godhani, Mr. Armandas Balcytis, Mr. Xuewen Wang, Mr. Philip Kinsella, Dr. Sepideh Mina, Dr. Martina Abrigo, Dr. Hannah Askew, Dr. Priyamvada Venugopalan, Mr. Racim Radjef, Ms. Litty Thekkekara, Ms. Elena Goi, Mr. Md Mamun and Mr. Ahmed Osman. Thank you for providing such a friendly and cheerful environment in the research labs and around the Swinburne premises.

I would like to acknowledge the huge support given to me by Dr. Sylvia Mackie, Dr. Brenton Hall, Ms. Tatiana Tchernova, Ms. Johanna Cooper and Ms. Susan Lloyd-Angol for different tasks throughout this entire period at Swinburne.

This PhD journey would not have been possible without the financial support received through the Vice Chancellors Research Scholarship from Swinburne University of Technology. Thanks again Swinburne, for giving me the opportunity to fulfil one of the prevalent dreams of my life.

I kindly remember the friends of Swinburne Optics and Photonics (SOAP). It was fun to spend the time with you guys over endless talks and games in our little gatherings. A big thank you to all my Swinburne friends from different Research Centres who are based in the AV building for saying hello to me every single day and sharing your stories over the walks to one end to the other end of Swinburne. We laughed together and cried together. That is because of “Finding the friends with the same mental disorder”. Priceless!

Especial thanks to SwinHealth including Mr. Josh Sasai and Dr. Srinath Jayasinghe for taking care of us when we were sick, particularly when our family members were thousands of miles away from us.

I am greatly indebted to my husband Rajith for standing by me from the day I met you. Thank you for believing in me and letting me to chase my dream. I could not have done this PhD without you. I do not have enough words to express my gratitude for what you have done and thank you for tolerating me and keeping our marriage going. It has been a blessing to be loved and cared by you, despite some arguments. viii

Many thanks to my parents and my two beautiful sisters Asha and Nirosha for their love and unwavering support shown upon me over the years. Thank you for believing in me. Life was very hard for me when you were all away but your constant encouragement has lifted me up.

To my gorgeous little nieces Amaya and Sinadi, how blessed I am to be surrounded by your love though I miss your big hugs every single day. Thank you for cheering me up with your sweet talks when the days were too hard to deal with.

Thanks to my two brother in-laws Nadeesh and Sanjeewa for taking care of my parents during the tough times. You guys were great and never let them feel alone.

I would like to thank my in-laws for their support and blessings during this PhD. Thank you for understanding me and my passion and for letting me to go with your son/brother to fulfil my dream. I know the past days would have been more pleasant to deal with if your lovely son/brother was around the corner. Thanks again for your sacrifice.

I take this opportunity to thank my teachers at Udugampola Primary School, Holy Cross College and academic staff at University of Sri Jayewardenepura, Sri Lanka. Thanks for guiding and shaping me to what I am today. I am forever grateful to have met you all in different phases of my life story.

Many thanks to all my friends and their family members and especial thanks goes to Suneetha, Mekhani, Anuththara and Samanthi for caring for us during the bright and dark days of the life in Melbourne and sharing your family stories with us to let us feel that we have a supportive family here.

Last but not least, I express my sincere gratitude and love to all the people here in Australia and back home in Sri Lanka, for your wonderful thoughts, support and helping hands given me to this date to make my dream come true. Thank you!

Technical Acknowledgements

The work included in this thesis would not have been possible to complete without the guidance and technical support of the following people.

Swinburne University of Technology

 Ms. Katharine Adcroft for chemical training and the technical support at ATC 901 laboratory.  Mr. Aditya Shah for Emitech coating unit training.  Dr. Daniel White for technical assistance with Emitech coating unit.  Dr. De Ming Zhu for Renishaw inVia Raman microscope training, profilometer training, technical support at ATC 803 laborartory and for XPS data collection.  Dr. James Wang for SEM imaing training and technical support at ATC basement laboratories.  Dr. Thomas Ameringer for J. A. Woollam ellipsometer training and technical assistance in AV laboratories.  Mr. Mark Kivinen for making all customised sample holders as per my requests at the CQOS workshop.  Dr. Karyn Jarvis for coordinating the technical issues in the ellipsometer with the J. A. Woollam company.  Prof. Shannon Notley and Dr. Hayden Webb for their guidance and tips to prepare samples for AFM imaging.  Dr. Nicholas Reynolds for his help in AFM imaging.  Ms. Yeannette Lizama for Secotom cutter training and the technical assistance at ATC basement labs.  Mr. Andrew Moore (Andy) for being my saviour whenever there was a technical issue in the Emitech coating unit and helping me to sandblast samples.  Ms. Pierrette Michaux and Mr. Xijun Li (Gordan) for clean room training.  Mr. Dan Kapsaskis for spectrophotometer training at AMDC 1001.  Ms. Savithri Galappathie for technical assiance in chemical laboratories.

RMIT University

 A/Prof. Sharath Sriram and Dr. Philipp Gutruf for helping with cross section SEM imaging.  Mr. Yuxun Cao for his help in sample cutting using DAD dicing saw.

Other

 Dr. Lachlan Hyde from Melbourne Centre for Nanofabricaton for the intial ellipsometry modelling training and technical assitance for issues associated with the ellipsometer.  Dr. Daniel Schmidt from National University of Singapore for his expertise knowledge and support for Mueller matrix ellipsometry, data modelling and collaborative research work on glass OAD86°.  Dr. Daniel Day and Mr. Derek Huxley from Warsash Scientific Pty. Ltd. for their technical assistance in Renishaw Raman microscope.  Ms. Olga Milikofu from Renishaw plc, UK for her instructions and guidance for Streamline Raman mapping.  Mr. Greg Pribil, Mr. Andrew Martin and Mr. Jeff Hale, Applications Engineers from J. A. Woollam Co., Inc, USA, for their eager help with technical issues of the ellipsometer and the CompleteESASE software.  Mr. Christian Gow from Coherent Scientific Pty. Ltd. for his instructions and help in AFM imaging.

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Declaration

I, Nilusha Perera declare that this thesis entitled:

“Study of Far-Side Geometrical Enhancement in Surface-Enhanced Raman Scattering” is my original work and contains no material which has been submitted previously, in whole or in part, in respect of any other academic award and to the best of my knowledge and belief contains no material previously published except where due reference is acknowledged in the text.

Mahamaluge Nilusha Mary Nilanthi Perera

Centre for Quantum and Optical Science Faculty of Science, Engineering and Technology Swinburne University of Technology Melbourne, Australia

Dated this day, 18th July 2017

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Nomenclature

AFM Atomic force microscopy AOI Angle of incidence ATR Attenuated total reflection CCD Charged-coupled device DDA Discrete-dipole approximation E. field Electric field EBL Electron beam lithography EF Enhancement factor EM Electromagnetic FDTD Finite-difference time-domain FEM Finite element method FTM Film thickness monitor FWHM Full-width half-maximum GE Generalised ellipsometry GEF Geometrical enhancement factor IR Infrared KK Kramers-Kronig K-R Kretschmann-Raether LSPR Localised surface plasmon resonance MSE Mean-squared error NA Numerical aperture NPs Nanoparticles OAD Oblique angle deposition OD Optical density PVD Physical vapour deposition SAM Self-assembled monolayer SD Standard deviation SE Spectroscopic/standard ellipsometry SEM Scanning electron microscopy SERS Surface-enhanced Raman scattering SPP Surface plasmon polariton SPR Surface plasmon resonance TE Transverse electric TM Transverse magnetic UV Ultra-violet Vis Visible XPS X-ray photoelectron spectroscopy

Contents

Dedication ...... iii

Abstract ...... v

Acknowledgements ...... vii

Technical Acknowledgements ...... xi

Declaration ...... xiii

Nomenclature ...... xv

Contents ...... xvii

List of Figures ...... xxi

List of Tables ...... xxxi

Chapter 1 ...... 1

1 Introduction ...... 1 1.1 Motivation ...... 1 1.2 Outline of the thesis ...... 3

Chapter 2 ...... 7

2 Literature Review ...... 7 2.1 Introduction to photon scattering ...... 7 2.2 Raman scattering ...... 9 2.3 Surface-enhanced Raman scattering (SERS) ...... 12 2.3.1 Localised surface plasmon resonance (LSPR)...... 17 2.3.2 Fabrication methods of SERS substrates...... 19 2.3.3 Applications of SERS ...... 23 2.4 Geometrically-enhanced SERS ...... 25 2.5 Dielectric function of metal ...... 30 2.6 Effective optical constants of metallic nanostructured films...... 32 2.7 Summary ...... 36

Chapter 3 ...... 37

3 Experimental Methods ...... 37 xvii

3.1 Sample fabrication ...... 37 3.1.1 Materials ...... 37 3.1.2 Oblique angle deposition (OAD) ...... 37 3.1.3 Emitech K975X turbo evaporator ...... 39 3.2 Sample imaging ...... 42 3.2.1 Scanning electron microscopy (SEM) ...... 42 3.2.2 Atomic force microscopy (AFM) ...... 43 3.3 Sample characterisation ...... 43 3.3.1 Optical profilometry ...... 43 3.3.2 Spectrophotometry ...... 44 3.3.3 Ellipsometry ...... 45 3.3.4 Raman spectroscopy ...... 46 3.3.5 X-ray photoelectron spectroscopy (XPS) ...... 50 3.4 Summary ...... 50

Chapter 4 ...... 51

4 Effective Optical Constants of Nanostructured Ag Films ...... 51 4.1 Introduction ...... 51 4.2 Determination of film thickness by profilometry ...... 52 4.3 Spectrophotometry of planar Ag films and Ag OAD films ...... 53 4.4 Image analysis of Ag OAD films ...... 55 4.5 Standard ellipsometry ...... 59 4.5.1 Theory...... 59 4.5.2 Results ...... 62 4.6 Anisotropy of Ag OAD86° ...... 66 4.7 Mueller matrix ellipsometry ...... 69 4.7.1 Theory...... 69 4.7.2 Results ...... 73 4.8 Conclusion ...... 89

Chapter 5 ...... 91

5 Theoretical Modelling of Geometrical SERS Enhancement ...... 91 5.1 Introduction ...... 91 5.2 Fresnel equations ...... 91 5.2.1 Parallel polarisation (p or TM polarisation) ...... 92 5.2.2 Perpendicular polarisation (s or TE polarisation) ...... 93 xviii

5.2.3 Absorbing medium ...... 95 5.3 Revisiting the current models ...... 97 5.3.1 The EM model by Funke and Wackerbarth ...... 97 5.3.2 The Fresnel model by Jayawardhana et al...... 99 5.4 Extended Fresnel model ...... 100 5.5 Conclusion ...... 107

Chapter 6 ...... 109

6 Experimental Study of Geometrical SERS Enhancement ...... 109 6.1 Introduction ...... 109 6.2 Raman experiments ...... 110 6.2.1 Raman signal dependence on solid angle ...... 110 6.2.2 Raman signal dependence on excitation laser power ...... 111 6.2.3 Raman depth-of-field ...... 113 6.2.4 Raman signal dependence on the source polarisation ...... 115 6.3 SERS experiments ...... 117 6.3.1 Repeatability of OAD fabrication ...... 117 6.3.2 SERS analytes ...... 118 6.3.3 SERS intensity dependence on the OAD angle ...... 122 6.3.4 SERS intensity dependence on the solid angle ...... 125 6.3.5 SERS depth-of-field ...... 126 6.3.6 SERS intensity dependence on the laser power ...... 128 6.4 Geometrical SERS experiments ...... 129 6.4.1 Streamline Raman mapping ...... 129 6.4.2 Wavelength dependence ...... 135 6.4.3 Substrate material dependence ...... 140 6.4.4 Angle of incidence dependence ...... 143 6.4.5 Polarisation dependence ...... 147 6.4.6 OAD layer thickness dependence ...... 151 6.5 XPS analysis of Ag OAD films ...... 155 6.6 Conclusion ...... 160

Chapter 7 ...... 163

7 Conclusion and Outlook ...... 163 7.1 Thesis conclusions ...... 163 7.2 Future work ...... 165 xix

References...... 169

Author’s Publications and Presentations ...... 197

List of Figures

Figure 1.1: Schematic of near-side and far-side illumination of the SERS substrate. Noble metal nanostructures are fabricated on transparent substrate and analytes are adsorbed to the nanostructures. Please note that schematic is not to scale...... 1

Figure 2.1: A Jablonski diagram (top) representing the molecular electronic and vibrational states and (a), (b) the simplified Jablonski diagrams of Rayleigh and Raman scattering and (c), (d) those two scattering processes in the quantum mechanical point of view with the excitation to a virtual state. EL and ES are the energy of incident and scattered photons, respectively. Adapted from [26]...... 8

Figure 2.2: Scheme of Stokes and anti-Stokes Raman scattering...... 10

Figure 2.3: Galactose and glucose hold the same chemical formula and extremely similar chemical structure, but generate two distinct Raman spectra. Adapted from [32]...... 11

Figure 2.4: SERS spectra of different biological species and an environmental pollutant detected in aqueous phase with analyte concentrations ranging from femtomolar to attomolar. Adapted from [41]...... 13

Figure 2.5: Schematic representation of the electromagnetic SERS process. Reproduced from [46]...... 14

Figure 2.6: Schematics of a) surface plasmon polaritons (SPP) and b) localised surface plasmon (LSP). Adapted from [54]...... 17

Figure 2.7: Size and shape dependent LSPR spectra of Ag nanostructured samples labelled as A-H with varying in plane width a and out of plane height b. Reproduced from [61]. .... 19

Figure 2.8: Dependence of Raman enhancement on excitation wavelength for different metals. Reproduced from [63]...... 20

Figure 2.9: Formation of hot spots in nanoparticles of monomers, dimers and trimers. The analyte trapped within the “hot” regions between nanoparticles exhibits a strong SERS effect due to the highly localised plasmon field. The strongest local field enhancement is observed in trimers with the excitation wavelength of 632.8 nm. Reproduced from [34]. 21

Figure 2.10: SEM images of various types of SERS active substrates fabricated by different techniques. Adapted from [10]...... 22

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Figure 2.11: Schematic of the near-side and far-side illumination of the SERS substrate. .. 25

Figure 2.12: Schematic of the incident beam path and the possible excitation of SPP at the metal-air interface for a) Kretschmann-Raether and b) Otto configurations...... 28

Figure 2.13: Schematic diagram of the transition from discrete electronic levels in isolated atoms to electronic bands in the solid for noble metals. Adapted from [104]...... 31

Figure 2.14: Derived effective optical constants of different metal layers deposited on glass a) refractive index and b) the extinction coefficient. Adapted from [123]...... 34

Figure 2.15: Uniaxial dielectric function of randomly distributed Ag nanoparticles with the optical constants of bulk Ag in solid blue line. The left-hand side axis shows the real part of the dielectric constant and the right-hand side shows the imaginary part. Adapted from [125]...... 35

Figure 3.1: Oblique angle deposition and formation of nanoislands. Adapted from [133]. 38

Figure 3.2: Photograph of the Emitech K975X turbo evaporator with parts labelled...... 39

Figure 3.3: The sketch for sample positioning inside the vacuum chamber and the tooling factor calculation...... 41

Figure 3.4: Schematic of the oblique angle deposition set up...... 42

Figure 3.5: Instrument set up of Cary 50 UV-Vis spectrophotometer. Adapted from [141]...... 44

Figure 3.6: Instrument set up of J. A. Woollam M-2000XI ellipsometer with focus probes. 46

Figure 3.7: Photographs of a) Renishaw inVia Raman spectroscope and b) a more detailed view of the optical setup used to guide light to the microscope and CCD...... 47

Figure 3.8: A typical SERS spectrum of thiophenol collected from Ag OAD86° 100 nm film with far-side excitation of the sample at 633 nm. The background was removed by applying a 2nd order spline with single weighting, smoothing parameter  =20,000 based on the algorithm published by [144]...... 49

Figure 4.1: Examples of step slides created on a) planar Ag film and b) Ag OAD86° film with a nominal thickness of 100 nm...... 52

Figure 4.2: The light transmission through OD1, microscope glass, planar Ag films and Ag OAD films measured with Cary 50 UV-Vis spectrophotometer. The Ag OAD films have a nominal thickness of 100 nm...... 54

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Figure 4.3: Absorbance spectra of Ag OAD70°, OAD80° and OAD86° films for a nominal thickness of 100 nm. The absorbance measurements were performed normal to the surface of the sample. Data were corrected for the absorption of the glass substrate...... 55

Figure 4.4: SEM images (left) of Ag OAD films deposited at a) 70°, b) 80° and c) 86° (scale bar 100 nm) and the corresponding ImageJ analysis results (right)...... 56

Figure 4.5: 2D view (left) and 3D view (right) of the AFM images of samples of Ag a) OAD70°, b) OAD80° and c) OAD86°...... 57

Figure 4.6: Box plots of the a) nanoparticle area and b) height calculated using the AFM images of OAD70°, OAD80° and OAD86° films by the NanoScope Analysis software...... 58

Figure 4.7: Representation of elliptically polarised light...... 60

Figure 4.8: Wavelength dependence of the refractive index as determined by the Cauchy layer for microscope glass and sapphire substrates. The corresponding constants of the Cauchy layer for glass and sapphire are shown on the graph...... 63

Figure 4.9: The effective optical constants of planar Ag 50 nm and 100 nm films as derived by the B-Spline model in CompleteEASE software and compared with the bulk silver values of Palik...... 64

Figure 4.10: Effective optical constants of Ag OAD70°, OAD80° and OAD86° films on glass substrates as determined by the B-Spline optical model...... 65

Figure 4.11: Cross-sectional SEM image of a Ag OAD86° 100 nm film. Vapour flux is from right to left during the oblique angle deposition...... 66

Figure 4.12: Absorbance spectra of Ag OAD86° 100 nm film for different rotations in the sample plane. The Xenon source polarisation was found to be along 30° with respect to the direction of the vapour flux and held fixed as the sample was rotated. The Xe source polarisation was measured with a polaroid filter. Repeatability of these measurements was generally better than 0.01 absorbance units...... 67

Figure 4.13: The effective optical constants of Ag OAD86° 100 nm modelled with a B-Spline layer based on standard ellipsometry measurements at different in-plane rotation angles. The insets show the direction of the vapour flux (black arrow) for the orthogonal standard ellipsometry measurements...... 67

Figure 4.14: Box plots of the a) nanoparticle length measured parallel and perpendicular to the direction of vapour flux, and b) the gap distance between nearest neighbours parallel and perpendicular to the direction of the vapour flux for Ag OAD86° 100 nm on glass...... 68

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Figure 4.15: Schematic showing the Euler angles (,,   ) and the orthogonal rotations as provided by rotational matrix A ...... 72

Figure 4.16: Experimental and best match generalised ellipsometry data versus sample rotation angle for AOI of 50°, 60° and 70° at  = 514 nm for Ag OAD86° 100 nm film deposited on glass...... 75

Figure 4.17: (a) Ordinary and (b) extraordinary dielectric constants of Ag OAD86° 100 nm deposited on glass. The results are based on a generalised oscillator layer representing three Gaussian oscillators and one Tanguy oscillator...... 77

Figure 4.18: a) The effective optical constants of the Ag OAD86° 100 nm film deposited on glass substrate in the sample coordinates of ( abc,, ) and b) the orientation of the uniaxial model. Laboratory coordinates are ( x,, y z ), where the sample plane is [ xy, ] and the plane of incidence is [ xz, ]...... 78

Figure 4.19: Box plots of the a) nanoparticle length measured parallel and perpendicular to the direction of the vapour flux and b) gap distance between nearest neighbours parallel and perpendicular to the direction of the vapour flux for Ag OAD86° 100 nm on sapphire...... 80

Figure 4.20: (a) Ordinary and (b) extraordinary dielectric constants of Ag OAD86° 100 nm deposited on sapphire. The results are based on a generalised oscillator layer representing three Gaussian oscillators and one Tanguy oscillator...... 81

Figure 4.21: Experimental and best match generalised ellipsometry data versus sample rotation angle for AOI of 50°, 60° and 70° at  = 514 nm for Ag OAD86° 100 nm film deposited on sapphire...... 82

Figure 4.22: The effective optical constants of the Ag OAD86° 100 nm film deposited on sapphire, in the sample coordinates ( ) as extracted from the uniaxial optical model.

...... 83

Figure 4.23: Average absorbance measurements of Ag OAD86° films on glass and sapphire normalised to [0,1], together with the corresponding refractive indices of the underlying substrates...... 84

Figure 4.24: The a) ordinary and b) extraordinary effective optical constants of the Ag OAD86° 100 nm film deposited on fused-silica substrate. Results are derived from Mueller matrix ellipsometry analysis of the near-side and far-side of sample...... 86

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Figure 4.25: The difference between the uniaxial optical constants for the near-side and the far-side measurements of the Ag OAD86° 100 nm film, plotted against the wavelength. ... 88

Figure 5.1: Representation of p polarisation. The sample plane is [ xy, ] and the plane of incidence is [ xz, ]...... 92

Figure 5.2: Representation of s polarisation. The sample plane is [ ] and the plane of incidence is [ ]...... 93

Figure 5.3: Schematic of light incident onto a plane surface of an absorbing medium and the light reflection from and transmission through the next boundary...... 95

Figure 5.4: Calculated electromagnetic enhancement plotted against wavelength for the nitrothiophenol vibrational mode at 1331 cm-1 for a) Ag and b) Au particles in air and quartz dielectric environments. Reproduced from [19]...... 98

Figure 5.5: Electric field intensity distribution in the yz plane cutting through the centre of the nanogap between two Ag nanoparticles for a) near-side and b) far-side excitation from FDTD modelling. The light is incident from below in this view. Reproduced from [20]...... 99

Figure 5.6: Schematic of a) near-side and b) far-side interrogation of the SERS substrate with the corresponding notations that are used in the extended Fresnel model...... 100

Figure 5.7: The wavelength-dependent near-side and far-side SERS electric field amplitudes calculated based on Equations (5.26) and (5.30) when the incident polarisation was aligned parallel and perpendicular to the ordinary axis of the uniaxial model of glass OAD86°. ...103

Figure 5.8: The average geometrical enhancement plotted against excitation wavelength at the film-substrate boundary and at the air-side of the substrate facing the collection lens...... 104

Figure 5.9: The wavelength-dependent average geometrical enhancement factor for far-side versus near-side excitation, calculated at the film-substrate interface by the extended Fresnel model...... 105

Figure 5.10: Angle dependence of the SERS field for near-side and far-side and the corresponding geometrical enhancement factor...... 106

Figure 6.1: Raman scattering signal dependence on the solid angle. The error bars mark the standard deviation (SD) of ten measurements at different locations of the Si wafer...... 111

Figure 6.2: Dependence of Si peak counts at 520 cm-1 on the excitation laser power. The dash line marks the line of best fit with slope ~1...... 112

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Figure 6.3: Comparison of Si peak intensity (corrected for the excitation laser power and acquisition time) with the excitation laser power for all objectives available on Renishaw system...... 112

Figure 6.4: Comparison of depth profiles of objectives including 5, 20, 50L, 50 and 100...... 113

Figure 6.5: Schematic of depth-of-field ranges of high and low NA objectives. Reproduced from https://www.microscopyu.com/microscopy-basics...... 114

Figure 6.6: Si peak intensity for normal and orthogonal polarisation states of the laser source plotted against the excitation wavelength...... 115

Figure 6.7: Reproducibility of Ag OAD86° 100 nm films...... 117

Figure 6.8: Molecular structure of thiophenol with colour codes of black (Carbon atoms), white (Hydrogen atoms) and yellow (Sulphur atom). Adapted from https://en.wikipedia.o rg/wiki/Thiophenol#/media/File:Thiophenol-3D-balls.png...... 118

Figure 6.9: Measured peak positions for Raman and SERS spectra of pure thiophenol and thiophenol SAM excited with 514 nm...... 119

Figure 6.10: Molecular structure of benzyl mercaptan with colour codes of black (C atoms), light grey (H atoms) and yellow (S atom). Adapted from https://en.wikipedia.org/wiki/Be nzyl_mercaptan#/media/File:Benzyl-mercaptan-3D-balls.png...... 121

Figure 6.11: Peak positions in the SERS spectrum of benzyl mercaptan SAM excited with 514 nm...... 122

Figure 6.12: Intensity of average thiophenol SERS spectra for six different Ag OAD films with a nominal thickness of 100 nm deposited at angles of 78°-88° using a custom-made holder (inset)...... 123

Figure 6.13: SERS intensity dependence on the vapour deposition angle...... 123

Figure 6.14: SEM images of Ag OAD films deposited at deposition angles of a) 78°, b) 80°, c) 82°, d) 84°, e) 86° and f) 88° with a nominal thickness of 100 nm...... 124

Figure 6.15: Average peak intensity of the four main thiophenol peaks plotted against the solid angle. The error bars represent the standard deviation of the measurements...... 126

Figure 6.16: Average peak intensity plotted against the sample displacement from the focal plane of the laser beam...... 127

xxvi

Figure 6.17: Average peak intensity of thiophenol peaks from the SERS spectra plotted against the excitation laser power of the sample. The error bars represent the standard deviation of six measurements on the sample...... 128

Figure 6.18: Schematic of SERS signal collection geometry of a) near-side and b) far-side of the Ag OAD film deposited on transparent substrate...... 129

Figure 6.19: SEM image of the Ag OAD86° 100 nm film, revealing island-like nanostructures...... 130

Figure 6.20: SERS counts of three main thiophenol peaks at 1000, 1021 and 1072 cm-1 recorded at a point on the selected area for the near-side during the Streamline mapping...... 130

Figure 6.21: Streamline Raman mapping of near-side of Ag OAD86° 100 nm. The step size was 20 μm and total scan area was 400  400 μm2. Note that the range of the colour scale is different for each map...... 131

Figure 6.22: Streamline Raman mapping of far-side of Ag OAD86° 100 nm. The step size was 20 μm and total scan area was 400  400 μm2. Note that the range of the colour scale is different for each map...... 132

Figure 6.23: Histogram of the baseline-to-peak heights of thiophenol SERS peaks of 1, 2 and 3 as labelled in Figure 6.20 and the total counts of peaks for the near-side (NS) and far-side (FS), respectively...... 133

Figure 6.24: Normalised counts of wavelength-dependent Raman spectra of pure thiophenol recorded using 5 lens in the near-side excitation geometry. The raw counts were normalised to [0, 1] using OriginPro 9.1...... 135

Figure 6.25: Normalised counts of Raman spectra of glass at 514 nm and 785 nm excitation using OriginPro 9.1...... 136

Figure 6.26: Normalised counts of wavelength-dependent thiophenol SERS spectra collected in the far-side excitation geometry using 5 lens. The raw counts have been normalised to [0, 1] and vertically offset for comparison...... 137

Figure 6.27: Average thiophenol peak intensities versus the excitation wavelength for near- side and far-side excitation geometries of Ag OAD86° 100 nm deposited on microscope glass. The error bars show the standard deviation of the ten measurements across the sample surface...... 138

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Figure 6.28: The experimental geometrical enhancement factors for Ag OAD86° 100 nm on glass as a function of excitation wavelength. The error bars show the standard deviation of the calculated geometrical enhancement factor...... 139

Figure 6.29: SEM image of Ag OAD86° 100 nm deposited on a sapphire substrate (scale bar 100 nm)...... 141

Figure 6.30: Average thiophenol peak intensity for Ag OAD86° 100 nm on sapphire sample plotted against the excitation wavelength. The error bars show the standard deviation of the ten measurements across the sample surface...... 141

Figure 6.31: The experimental geometrical enhancement factors for Ag OAD86° 100 nm on sapphire as a function of excitation wavelength. The error bars show the standard deviation of calculated geometrical enhancement factor...... 142

Figure 6.32: Normalised averaged absorbance spectra of Ag OAD86° 100 nm deposited on glass and sapphire. The excitation laser wavelengths are marked by dashed lines...... 143

Figure 6.33: Schematic of the near-side excitation geometry for the Ag OAD86° film with varying angles of incidence i ...... 144

Figure 6.34: Average peak intensity for near-side and far-side excitation plotted against the angle of incidence ranging from 0-85°. The error bars show the standard deviation of two measurements...... 144

Figure 6.35: Geometrical enhancement dependence on the angle of incidence for Ag OAD86° 100 nm deposited on sapphire substrate. The error bars show the standard deviation of the calculated geometrical enhancement factor...... 146

Figure 6.36: Average counts of thiophenol SERS spectra corrected for laser power and acquisition time for excitation of the extraordinary and ordinary axes of the sample. Both near-side and far-side geometries are presented as labelled...... 148

Figure 6.37: Average counts of benzyl mercaptan SERS spectra corrected for laser power and acquisition time for excitation of the extraordinary and ordinary axes of the sample. Both near-side and far-side geometries are presented as labelled...... 148

Figure 6.38: Polar plot of the average peak intensity of benzyl mercaptan SERS spectra for unpolarised SERS (without PF) and polarised SERS (with PF). The average peak intensity has been plotted against the in-plane sample rotation angle with respect to the fixed polarisation direction of the incident polarisation light...... 150

xxviii

Figure 6.39: SEM images of Ag OAD86° films with nominal thicknesses of a) 100 nm, b) 150 nm c) 200 nm deposited on glass substrates using oblique angle deposition, and d) box plots representing the nanoparticle diameter as determined by the ImageJ analysis...... 152

Figure 6.40: Polarised absorption spectra of Ag OAD86° films with a nominal thickness of 100 nm, 150 nm and 200 nm measured along two orthogonal directions with respect to the deposition vapour flux...... 153

Figure 6.41: Average peak counts plotted against the nominal thickness of the Ag OAD layer for the excitation of the extraordinary and ordinary axes of the sample in the near-side (NS) and far-side (FS) geometries...... 154

Figure 6.42: The XPS survey spectra of uncoated a) glass, b) sapphire and c) Si substrates...... 155

Figure 6.43: The XPS survey spectra of a) Ag OAD86° and b) Ag OAD86° treated with thiophenol ethanolic solution. Both films were deposited on microscope glass substrates and have a nominal thickness of 100 nm...... 157

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List of Tables

Table 2.1: Energy change and classification corresponding to visible light scattering processes. Scattering cross section per molecule is shown in cm2 [8, 25, 28]...... 9

Table 2.2: Summary of the average enhancement factors reported for SERS substrates fabricated by different fabrication techniques as discussed in [10]...... 16

Table 2.3: A brief summary of previously reported applications of SERS in the fields of chemical and biomolecular sensing...... 24

Table 3.1: List of materials used for the sample fabrication and analysis in this Chapter... 37

Table 3.2: Laser-grating combinations of Renishaw inVia Raman microscope. The available gratings are represented by the number of grooves per mm (l/mm). Streamline refers to a mapping function that will later be used in this thesis. The excitation wavelengths used here are mentioned as commercially-labelled wavelengths in the Renishaw inVia Raman system...... 48

Table 3.3: List of objectives attached to the Renishaw inVia Raman microscope...... 48

Table 4.1: Effective film thickness of the Ag OAD86° 100 nm film corresponding to the best match model for four sample plane rotation angles in 90° steps...... 66

Table 4.2: Summary of Mueller matrix analysis best match model results of Ag OAD86° 100 nm films deposited on substrates of fused-silica, glass and sapphire, respectively...... 88

Table 6.1: Polarisation-dependent Raman peak intensity for Si at 520 cm-1...... 116

Table 6.2: Comparison between normal Raman peaks and SERS peaks of thiophenol...... 119

Table 6.3: Analysis of the wavelength dependence of Raman peaks of Si and thiophenol, and SERS peaks of thiophenol. The peak positions and the FWHM of peaks were determined with a Gaussian fit...... 120

Table 6.4: Experimental SERS peaks of benzyl mercaptan [232, 233]...... 121

Table 6.5: Image analysis results for Ag OAD samples fabricated using the customised holder for angles from 78° to 88°...... 125

Table 6.6: Wavelength-dependent dielectric function of Ag OAD86° 100 nm on glass extracted from the uniaxial model...... 139

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Table 6.7: Wavelength-dependent dielectric function of Ag OAD86° 100 nm on sapphire extracted from the uniaxial model...... 141

Table 6.8: The peaks of XPS survey spectra and the corresponding surface atomic composition of glass, sapphire and Si substrates. Three analyses were performed on each substrate to calculate the mean ± SD in % atomic composition...... 156

Table 6.9: The atomic composition and the relative elemental ratios for Ag OAD86° films deposited on glass, sapphire and Si substrates...... 159

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Chapter 1

1 Introduction

1.1 Motivation

Recent advances in fabrication techniques [1-5] have expanded the potential applications of SERS sensing, especially in the fields of chemical and biomolecular sensing [6-10]. Direct excitation of analytes that are chemically or physically adsorbed to the substrate has attracted the attention of many SERS research groups [11-13]. Over the past 30 years, several groups have shown an increase of the SERS signal with different nanostructures fabricated with noble metals on transparent substrates when the sample is illuminated in the far-side geometry compared to the conventional direct illumination [14-21]. The far- side geometry describes the situation where laser light first penetrates the transparent substrate and then reaches the metal film and analytes as shown in Figure 1.1.

However, the origin of this geometrically-enhanced SERS signal has not been satisfactorily explained until the recent work carried out by two independent groups in 2013. Funke and Wackerbarth [19] used a theoretical model based on the electromagnetic enhancement whereas Jayawardhana et al. [20] used an analytical model based on the Fresnel equations to validate the experimentally observed far-side enhanced SERS signal. Interestingly, the explanations provided by both groups tend to explain the increased SERS signal Page | 1

Chapter 1

qualitatively within their experimental limits. The proposed models are extensively discussed in Section 5.3. However, a detailed quantitative study of the far-side geometrically-enhanced SERS signal has not yet been undertaken by any group, to our knowledge. Therefore, in order to identify the key contributing factors of this geometrically- enhanced SERS signal and considering the gaps in our current understanding, two main objectives were defined in this thesis.

The two main objectives of this thesis are:

1. To determine the effective optical constants of nanostructured thin Ag films and use those effective optical constants to theoretically model the far-side geometrical enhancement in the SERS signal. Conventional modelling methods such as finite-difference time-domain (FDTD) generally use the bulk properties of the material to generate the field intensities around nanoscale plasmonic structures. While the local fields help to understand some aspects of these plasmonic films, such as ‘hot spots’, they do not provide a convenient description of the average macroscopic behaviour. In contrast, ellipsometric measurements provide a spatial average of thin films and thus their effective optical constants as determined by an optical model that is used to represent the nominal structure of the sample. This allows us to use the effective dielectric constants of the nanostructured Ag films to model the geometrically- enhanced SERS signal and extend the current analytical model proposed by Jayawardhana et al. [20], with the inclusion of the absorbing metallic film for the corresponding electric field calculations. Even though, Funke and Wackerbarth [19] used the size-dependent complex dielectric function of a metallic nanoparticle described by the Drude model at a single wavelength, there remains a need to apply an effective medium theory to represent the distribution of the nanoparticle sizes, shapes and the inter particle effects over a range of excitation wavelengths. Therefore, within the scope of this thesis, ellipsometry was used to accurately determine the effective optical constants of the samples under investigation.

2. To experimentally study the far-side geometrical enhancement in the SERS signal. The experimental results related to the geometrically-enhanced SERS signal found in the literature were very limited and restricted mainly to a given substrate at a particular excitation wavelength. According to our knowledge, the far-side excitation geometry has not been experimentally tested for different excitation wavelengths before. The aim of this experimental study is to perform a comparative study for the near-side and far-side SERS signal dependence on excitation wavelength, substrate material on which the

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Chapter 1

nanostructures are deposited and angle of incidence. This will also allow a comparison between the theoretical predictions of the extended Fresnel model and the laboratory results. As an extension to the work previously done by Jayawardhana et al. [20], a Ag OAD86° film with a nominal thickness of 100 nm fabricated using oblique angle deposition was used as the SERS-active substrate. The samples fabricated on transparent glass and sapphire dielectric substrates were used for the SERS experiments with thiophenol as the analyte. These experiments help to understand the key contributing factors in the geometrical enhancement, which is defined as the ratio of far-side to near-side SERS intensity.

Compared to the available resources found in the literature, the work presented in this thesis aims to investigate the key factors that might contribute to the geometrically- enhanced SERS signal, working from a more detailed theoretical foundation together with more rigorous experimental results. These objectives will lead to improve the SERS detection modes at the far-end of optical fibre probes and through process windows [22- 24], especially when accompanied by other near-field interaction effects.

1.2 Outline of the thesis

The research conducted to fulfil the requirements of this thesis entitled “Study of Far-Side Geometrical Enhancement in Surface-Enhanced Raman Scattering” is extensively discussed in the following chapters.

Chapter 2 presents the background information that is needed for the scope of work included in this thesis. The literature review begins by introducing the subject of photon scattering, followed by Raman scattering, surface-enhanced Raman scattering, geometrically-enhanced SERS, the dielectric function of metals and finally the effective optical constants of metallic nanostructured films as determined by ellipsometry. This brief review highlights the gaps in our current understanding of the geometrically-enhanced SERS signal and demonstrates the importance of a further study of the effect for future progress in terms of fibre-based applications in sensing and measurements of chemical processes through observation windows.

Chapter 3 presents the materials and experimental methods that were used in for the experimental work included in this thesis. The sample fabrication technique known as oblique angle deposition is introduced. The experimental methods include sample imaging

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Chapter 1

by scanning electron microscopy (SEM) and atomic force microscopy (AFM), and sample characterisation based on optical profilometry, spectrophotometry, ellipsometry, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The corresponding experimental parameters are further discussed in each section.

Chapter 4 presents the ellipsometry work performed on Ag films based on both standard and generalised ellipsometry techniques. Prior to the ellipsometry analysis, the thin Ag films are analysed with profilometry and spectrophotometry. In the ellipsometry analysis, firstly, standard ellipsometry is used to identify the physical shape of the wavelength-dependent effective optical constants of Ag films and then generalised ellipsometry with Mueller matrix analysis is used to determine the effective optical constants of Ag OAD86° 100 nm films based on the observed anisotropy. The Ag OAD86° 100 nm films deposited on transparent glass, sapphire and fused silica substrates are used to investigate the wavelength-dependent optical constants of the films. The wavelength-dependent complex optical constants of the nanostructured Ag plasmonic film are extracted by a uniaxial optical model. Finally, the near-side and far-side optical constants of Ag OAD86° 100 nm are measured and compared.

Chapter 5 presents the theoretical models that are used to represent the geometrically- enhanced SERS signal. Firstly, the Fresnel equations are introduced for non-absorbing and absorbing media. Prior to presenting the current extended Fresnel model to theoretically confirm the geometrically-enhanced SERS, the previous models published by Funke and Wackerbarth [19], and Jayawardhana et al. [20] are presented. The limitations and the gaps in the previously proposed models are addressed by the extended Fresnel model with wavelength-dependent complex refractive indices of the nanostructured Ag film as determined by Mueller matrix ellipsometry. The underlying substrate material and the angle of incidence dependence in the geometrical enhancement factor are further discussed.

Chapter 6 presents a sequence of fundamental Raman and SERS experiments, together with a set of experiments to study the geometrically-enhanced SERS signal. The fundamental Raman and SERS experiments include the signal dependence on solid angle, excitation laser power, depth-of-field and source polarisation. These measurements provide an understanding of the “baseline” performance characteristics of the spectroscope. The Ag OAD86° film with a nominal thickness of 100 nm functionalised with thiophenol is used as a standard SERS substrate. A Streamline Raman mapping of the near-side and far-side of the sample is used to discuss the variability of the SERS signal and the geometrical enhancement factor across the sample. The other experiments discussed in this chapter cover the

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Chapter 1

dependence of the geometrically-enhanced SERS signal on the excitation wavelength, substrate material, angle of incidence and metal layer thickness. Furthermore, the extended Fresnel model predictions for the geometrical SERS enhancement are compared against the experimental results. Finally, XPS is used to examine and determine the chemical composition of the SERS substrates used in the work of this thesis.

Chapter 7 summarises the key findings of the work of this thesis and some perspective for future work.

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Chapter 2

2 Literature Review

2.1 Introduction to photon scattering

In light matter interaction, scattering is a fundamental optical phenomenon that occurs due to the spontaneous absorption of an incident photon and emission of another photon. The emitted photon is called the scattered photon and its energy compared to the initial energy of the incident photon is used to describe the elastic and inelastic scattering processes. Some of the common spectroscopic units that are used to represent the energy of a photon can be found in the forms of energy E [J] or [eV], angular frequency  [rad s-1], frequency  [Hz or s-1], wavelength  [m] or absolute wavenumber  [m-1]. These parameters are all related to each other by the equation:

hc E h     hc  , (2.1) 

where h  6.6310−34 J s is Planck’s constant and c  3108 m s−1 is the speed of light in a vacuum. The total energy of a molecule is a combination of electronic, vibrational, rotational and translational (thermal) energy. In spectroscopy, a molecule is described by a group of N number of nuclei and M number of electrons having 3(N+M) degrees of freedom to its own rotational motion. However, it has been shown that there are only 3N-5 and 3N-6 of vibrational degrees of freedom for linear and non-linear molecular systems, respectively [25]. The normal modes of molecular vibration are symmetric and asymmetric stretching, wagging, twisting, scissoring and rocking.

Interaction of light with a molecule can be represented by a Jablonski diagram with its energy levels. Figure 2.1 (top) is the schematic of a Jablonski diagram indicating electronic

states of a molecule (bold curves of S0 and S1) and rotational, vibrational states (thinner

lines of vi, i=0, 1, 2…) including possible energy transitions of radiative (dotted arrows) and

non-radiative (solid arrows) transitions. In the ground electronic state (S0), the electrons of the molecule occupy the lowest energy state allowed by the Pauli exclusion principle and

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Chapter 2

absorption of photons excites them into the transition of higher electronic or vibrational states.

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Chapter 2

As can be seen in Figure 2.1, infrared (IR) absorption is a one-photon event where the energy of the photon corresponds to the energy gap of the two vibrational states. The only possible way to reach the ground state is via non-radiative processes. As the energy is completely absorbed, no scattering occurs. In order to reveal the molecular information, the transmittance or reflectance spectrum of the molecules is recorded as the IR spectrum over a range of infrared frequencies. The energy bands of the spectra directly indicate the vibrational states of the molecules [27].

Rayleigh scattering (Figure 2.1 a and c) and Raman scattering (Figure 2.1 b and d) are two- photon events that occur with higher energy photons. When a photon interacts with a molecule, it is excited to a virtual state as shown in Figure 2.1 c and d. In most cases, it will relax back to the ground state. This process is known as the Rayleigh scattering and the frequency of incident and scattered radiation remain same. The scattering intensity is inversely proportional to the fourth power of the incident wavelength [27]. Table 2.1 shows a summary of visible light scattering processes and their corresponding properties.

Transition energy Name of category Property of scattering Scattering cross section (cm2) <10-5 cm-1 ~0 Rayleigh Elastic 10-26 10-5 cm-1 ~1 Brillouin Inelastic Raman 10-29 >1 cm-1 Inelastic SERS 10-16

Table 2.1: Energy change and classification corresponding to visible light scattering processes. Scattering cross section per molecule is shown in cm2 [8, 25, 28].

However, in some cases the scattering becomes inelastic, as there is a frequency shift between incident and scattered photons. The possibility of changing the relaxed state to a different vibrational energy level compared to the initial state is known as Raman scattering [27].

2.2 Raman scattering

Raman scattering was first observed by C.V. Raman in 1928 [29] and is used to identify the materials in any phase of solid, liquid or gas such as chemical or biological substances and human tissues. Raman scattering depends upon the change in the polarisability of the molecule with respect to its vibrational motion. Raman intensity is directly proportional to

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Chapter 2

the induced dipole moment ind of the molecule and can be written as the product of Raman polarisability  and the magnitude of the incident electromagnetic field Ei [30]:

222 IERaman ind i . (2.2)

When the final vibrational state is at a higher energy level than the initial state, the scattered photon has a lower energy than the incident photon and is called Stokes scattering. When the scattered photon relaxes to a lower energy state than the initial vibrational state of the molecule, it has a higher energy and this process is named anti-Stokes scattering. Anti- Stokes scattering is weaker than Stokes scattering at room temperature due to the relatively smaller population in the excited state (S1) of the molecules. The schematic of the two Raman scattering processes is depicted in Figure 2.2 and the relevant energy relationships for Stokes and anti-Stokes scattering are given in Equations (2.3) and (2.4).

Figure 2.2: Scheme of Stokes and anti-Stokes Raman scattering.

Stokes scattering: EEES L   vibration , (2.3)

Anti-Stokes scattering: EEES L   vibration , (2.4) where EL is the energy of the incident photon and ES is the corresponding energy of the scattered photon. Evibration represents the energy difference that occurred during the scattering process:

Ehvibration  L  S . (2.5)

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Chapter 2

The energy of the vibration mode can be calculated using Equation (2.5) when  L is the frequency of incident radiation and  S is the frequency of the scattered photon.

The wavelength shift  is responsible for the peaks in the Raman spectrum which represent the different vibrational modes of the molecules and is given by

7  1 1 10 nm  cm 1    . (2.6)  cm L nm  S nm 

Every polarisable molecule has a unique Raman spectrum, which can therefore be thought of as the fingerprint of the molecule. Figure 2.3 shows the unique Raman spectra of galactose and glucose molecules that have the same chemical formula but different structures. Raman spectroscopy is used to study the molecular vibrational modes such as bending, stretching and wagging. Most molecules in a sample have a very weak Raman signal, which is only ~10-10 times the intensity of the incident light. The signal becomes even weaker in cases with a monolayer or sub monolayer adsorbed species on the surface [31].

Figure 2.3: Galactose and glucose hold the same chemical formula and extremely similar chemical structure, but generate two distinct Raman spectra. Adapted from [32].

The total power of the Raman scattered light PRS scales linearly with the intensity of the excitation laser light I :  L 

P NI  , RS  L  RS (2.7) where N is the number of molecules in the laser excitation spot and  RS is the corresponding Raman cross section. In general, for Raman signal analysis, high intensity

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Chapter 2

light sources and high sensitivity detectors are commonly used, as the total power of the Raman scattered light is always very small. Surface-enhanced Raman scattering is often used to enhance the Raman signal in order to overcome the inherently weak Raman signal.

2.3 Surface-enhanced Raman scattering (SERS)

Surface-enhanced Raman scattering is a surface sensitive technique for overcoming the inherently weak Raman signal in a molecule. SERS enhances the normally weak Raman signal by up to 9-12 orders of magnitude if the analyte is adsorbed on a nanostructured metal film [33, 34]. The SERS spectrum of pyridine adsorbed on an electrochemically roughened silver electrode was first reported by Fleischmann et al. in 1974 [35]. They postulated that the higher signal intensity observed was due to an increased number of excited molecules residing on the enlarged surface area due to roughening. A few years later, Albrecht et al. concluded that the million fold enhancement originated from a surface effect which significantly increases the molecular Raman cross section [36]. In 1977 and 1978, Jeanmaire et al. and Moskovits independently proposed that the increased Raman intensity is the result of a complex interaction between the surface plasmon field excited on the metal and the molecules [37, 38].

Similarly to Raman scattering, the total power of SERS scattered light is directly proportional to the excitation of the incident laser light but is also enhanced by an additional factor M EM as shown in Equation (2.8) [39]. A detailed calculation of the total power of SERS is found elsewhere [8, 40].

P NI M . SERS  L  SERS EM (2.8)

The early observations and explanations of the SERS phenomenon have inspired both experimental and theoretical work on SERS in fields as diverse as chemistry, physics, surface science, biology and nanoscience. In the meantime, work on SERS has demonstrated its power as an analytical tool for molecular detection. Researchers have used SERS-active substrates as platforms for the detection of target species including dyes, biomarkers, drugs, pollutants, explosives, biomolecules, peptides, proteins, bacteria, viruses and blood cells [9, 10]. Analysing multiple-phases including liquid, organic and air samples has been of great interest for potential applications in environmental monitoring, national security and analytical chemistry, while reaching the sensitivity of the molecular concentrations ranging

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Chapter 2

from pM down to aM [41, 42]. Figure 2.4 shows the SERS spectra of DNA bases of thymine and adenine, bovine serum albumin (BSA) protein and an environmental contaminant of bis(2-ethyl-hexyl) phthalate detected at femto to atto molar concentrations.

Figure 2.4: SERS spectra of different biological species and an environmental pollutant detected in aqueous phase with analyte concentrations ranging from femtomolar to attomolar. Adapted from [41].

In order to explain the enhancement in SERS, two key mechanisms have been proposed: a) the electromagnetic enhancement (EM) and b) the chemical enhancement (CE). The EM enhancement is due to the excitation of the localised surface plasmon resonance (LSPR) of the nanostructured metal substrate [33]. The LSPR is an optically driven phenomenon that occurs due to the interaction between the incident light and the collective oscillation of conduction electrons in metals such as gold, silver, copper and aluminium [26, 43, 44]. It has been shown that the EM mechanism plays a dominant role in SERS [17, 45]. The total enhancement in the EM mechanism is well described by Equation (2.9), as a product of field intensities at incident light (excitation) and scattered light (emission) wavelengths coupled to the LSPR [8, 46]. Page | 13

Chapter 2

2 2 Eloc L  Eloc S  22 M EM MM12L  S  , (2.9) EEi L  i S  where Eloc and Ei are the localised and incident EM field amplitudes and L and S are the excitation laser and the scattered Raman wavelengths, respectively. The first EM enhancement M1  of the scattering occurs due to coupling of the incident light with the surface plasmons. Similarly, the second enhancement M 2  occurs when the Raman signal is coupled to the plasmon. A schematic representation of the electromagnetic SERS process is shown in Figure 2.5 followed by a summary of the steps involved during the enhancement.

Figure 2.5: Schematic representation of the electromagnetic SERS process. Reproduced from [46].

The steps involved in the EM enhancement in SERS can be described as;

1. A metallic nanoparticle with permittivity metal is surrounded by a medium with permittivity dielectric and a molecule is at the vicinity of the nanoparticle.

2. The nanoparticle is excited by the incident beam at the excitation wavelength and induces a local enhanced field around the nanoparticle.

3. The molecule is then excited by the enhanced local field and scatters the Raman signal in every direction.

4. The scattering process occurs due to the polarisability  of the molecule. As the locally enhanced field polarises the molecule, it produces a scattered field at the Raman wavelength. Page | 14

Chapter 2

5. Finally, the field scattered by the molecule also couples to the plasmon field around the nanoparticle and starts to re-radiate the so called “surface-enhanced Raman signal”.

It is commonly approximated that the SERS enhancement is the fourth power of the locally

4 enhanced electromagnetic field ( E ) produced by the metallic nanoparticles, assuming that incident and the Raman wavelengths are close enough [30]. However, the two wavelengths can be separated by several tens of nanometres and this wavelength shift plays a key role in the SERS process [46]. The enhanced fields are surface specific and decay rapidly with distance away from the surface [47]. Although the electromagnetic enhancement does not require the molecule to be in direct contact with the nanostructures, Baker and Moore [8] have introduced the distance-dependent EM enhancement for a molecule located at a distance d away from a nanoparticle with a radius a :

12 22a MMMEM  12L  S  . (2.10) ad

The EM enhancement is dependent on the structural geometry of the nanoparticle substrate, excitation wavelength, surrounding medium and the polarisation state of the excitation [33]. By controlling the size, shape, composition and the inter particle spacing of the nanoparticles structures, the LSPR can be tuned to achieve the optimum SERS signal at a particular wavelength of interest [31].

The chemical enhancement is involved when the analyte molecule forms a chemical bond (charge transfer complexes) with the nanostructured metallic surface [17, 33]. Due to the increased electron density in the molecule, the interactions with the excitation photons become much higher, resulting in the Raman signal increasing by an additional one or two orders of magnitude [33]. However, the chemical enhancement is a short-range effect which arises only on the molecular length scale [31].

The SERS enhancement factor is used as the figure of merit to compare different SERS substrates and introducing a simple definition is impossible due to the complexity of different experimental approaches. There are some disparities in the current definitions of the SERS enhancement factor (EF) due to the diversity of experimental situations and limitations such as single molecule, multiple molecules, spatial distribution of molecule and orientation of probe. Some of the common definitions used in the literature are single molecule enhancement factor (SMEF), analytical enhancement factor (AEF) and SERS substrate enhancement factor (SSEF). A comprehensive discussion of the SERS

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Chapter 2

enhancement factor can be found in [13]. Therefore, in favour of the substrate point of view, the most widely used definition for the average SERS EF evaluated at a single excitation wavelength can be calculated by

INSERS surf EF  , (2.11) INNRS vol where ISERS is the SERS intensity, Nsurf is the number of molecules bound to the metal substrate in the laser focal spot, INRS is the normal Raman intensity and Nvol is the number of molecules in the excitation volume [30]. This definition has been widely used to compare the average SERS enhancement factors across different SERS substrates used by different research groups. An example of a detailed approach of measuring enhancement factor is discussed by McFarland et al. [48] considering the number of benzenethiol molecules probed in a liquid sample and on a SERS substrate. A brief summary of average enhancement factors reported in the literature for different types of SERS substrates are listed in Table 2.2 as discussed in the review paper by Luo et al. [10]. The fabrication techniques are discussed later in more detail at Section 2.3.2.

Fabrication technique EF, metal type

Bottom up approach

Nanoparticle immobilisation 106, Au Oblique angle deposition (OAD) 108-1010, Ag Top down approach

Electron beam lithography (EBL) 108, Au Focused ion beam (FIB) 107, Au Ar ion sputtering 1010, Ag Metal induced chemical etching 107, Au Template-assisted

Anodic aluminium oxide (AAO) 109, Au Carbon nanotube 107, Au Colloid assembly 108, Ag ZnO nanowires 106, Au Combined techniques

EBL + Au reduction 108, Au EBL + particle binding 108, Au UV lithography + OAD 106, Ag

Table 2.2: Summary of the average enhancement factors reported for SERS substrates fabricated by different fabrication techniques as discussed in [10].

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Chapter 2

2.3.1 Localised surface plasmon resonance (LSPR)

Surface plasmons are a collective excitation of conduction electrons at the surface of noble metals and are the fundamental origin of plasmonics, where light-matter interaction at the nanoscale. The main applications of plasmonics are found in the fields of surface-enhanced spectroscopies [3, 49, 50] and fabrication of nanoscale optical and photonics devices such as waveguides, switches, super lenses and information storage devices [51-53]. The main two types of surface plasmons are surface plasmon polaritons (SPP) and localised surface plasmons (LSP) as graphically shown in Figure 2.6.

Figure 2.6: Schematics of a) surface plasmon polaritons (SPP) and b) localised surface plasmon (LSP). Adapted from [54].

The former arises when electromagnetic excitations (surface plasmons) are propagating at the dielectric-metal interface of thin continuous metallic films. However, SPP are evanescently confined in the direction perpendicular to their direction of propagation [55].

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With recent advances in fabrication of well-controlled nanostructures, the latter mechanism originates as a result of light interaction with nanoparticles (NPs) much smaller than the wavelength of the incident radiation [56]. Excitation of LSP results in strong scattering at a resonance wavelength and is responsible for the intense absorption peak. This absorption peak is generally referred to as the localised surface plasmon resonance (LSPR) maximum and is highly sensitive to the material used to fabricate the nanostructures, the size and shape of the nanostructures, size distribution of the NPs and the surrounding dielectric medium [54].

Plasmon spectroscopy is commonly used as an ultrasensitive detection platform, especially for chemical and biological molecules [43, 57]. However, the main interests of this section are limited to the theory and contribution of the LSPR to SERS. The extinction spectrum that includes both light absorption and elastic scattering of a metal sphere with a dielectric constant of metal  ri  i in a dielectric medium medium can be represented by

 2423Na 32   Extinction   medium i ,   2 (2.12)  ln 10       2  r   medium i   where a is the radius of the NP, N is the finite polarisable elements in the metallic NP that can interact with the applied electric field and  is the geometrical factor.

The factor accounts for the shape of the nanoparticles and it has been shown that can be varied by as much as 20 for nanoparticles with high aspect ratios. Generally, is considered equal to two for a sphere. The LSPR is sensitive to any small changes in the aspect ratio of ellipsoidal NPs and turns out to provide strong, polarisation-dependent spectral shifts and an increase in the intensity of the LSPR peak is reported as a result of an increase in the NP symmetry [56, 58]. Meanwhile an increase in the sharpness of the NP edges leads to a red- shift in the extinction due to increased charge separation. A red-shift of 47 nm was presented when the particle diameter was increased from 10 to 100 nm [43]. The surface plasmon resonance or the Mie resonance peak in the extinction spectrum is theoretically expected when r    2 medium [43]. For the most commonly used noble metals of Ag and Au, the LSPR is found in the visible range of the spectrum due to interband electronic transitions from the valence band to the conduction band with Ag exhibiting the strongest and sharpest band [59, 60]. Figure 2.7 shows the size and shape dependent LSPR peaks for nanoparticles fabricated by nanosphere lithography.

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The changes in the plasmon resonance peak position due to the adsorption of an analyte can be mathematically presented by

max m  n1  exp  2 d ld  , (2.13) where m is the bulk refractive index of the NP material, n is the change of refractive index induced by changes in the surrounding medium due to the presence of the analyte, d is the thickness of the analyte layer and ld is the EM field decay length [54]. The extinction peak is red-shifted when organic molecules that have higher refractive indices than the solvents and air are adsorbed on the NPs [43].

Figure 2.7: Size and shape dependent LSPR spectra of Ag nanostructured samples labelled as A-H with varying in plane width a and out of plane height b. Reproduced from [61].

Review articles by Petryayeva et al. and Hutter et al. have provided detailed discussions on fabrication techniques that have been commonly used for preparation of LSPR enabled substrates, including the methods of colloidal deposition, electro deposition, vacuum deposition, electron beam lithography and laser ablation [43, 56].

2.3.2 Fabrication methods of SERS substrates

Comparison of various SERS substrates is challenging due to their complexity. However, the ultimate goal of each fabrication method is to design a robust SERS substrate to achieve

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strong light localisation. In order to produce an ideal SERS substrate, the requirements of high repeatability within the sample and between batches, and high SERS activity that leads to high sensitivity and stability should be satisfied [31]. From a commercial point of view, it is important to have a reproducible substrate at a minimum cost with long shelf life.

The search for the ideal SERS substrate is an ongoing effort with various methods being reported in the literature over many years, beginning with the roughened silver electrodes used in the initial studies. It is important to control all of the factors that influence the LSPR in order to ensure the maximum SERS signal and the highest reproducibility. Material, dielectric environment, particle size, particle shape and inter-particle spacing are the major factors affecting the reproducibility of excitation of the LSPR by incident laser light. Ag, Au and Cu have mostly been used for substrate fabrication, as all three metals have LSPRs in the visible wavelength range, which makes them suitable for practical applications. Recent work is also exploring Al to exploit potential of the deep UV region for sensing [44]. The quality factor (Q ) determines the local field enhancement near plasmonic nanoparticles and depends on the relationship between the real and imaginary parts of the dielectric function of metal at the excitation wavelength, [62] as shown in Equation (2.14). It should be noted that this equation can only be applied to materials whose permittivity is precisely described by the Drude model. This results in the noble metals being the best enhancers in the visible range, with silver being more enhancing than gold [47] as indicated in Figure 2.8.

Re metal    Q r . (2.14)  Im metal   i

Figure 2.8: Dependence of Raman enhancement on excitation wavelength for different metals. Reproduced from [63]. Page | 20

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Preparation methods for SERS substrates have been improving ever since the first SERS observation. Many complex methods of fabricating SERS substrates have been reported in the literature and additional details can be found elsewhere [31, 64-66]. The two general methods of creating SERS active nanoparticles/ nanostructures are based on solution synthesis and nanofabrication. Aggregation of NPs with a target species is a direct technique, which provides a high SERS signal in the presence of hot spots. The main drawback in this method is the difficulty of controlling the formation of hot spots [10].

Researchers have investigated the formation of “hot spots”, where small regions contain the highly enhanced electric field that leads to high SERS sensitivity. These hot spots occur at nanometre-sized gaps and allow the SERS signal to be detected at a single molecule level. When two nanoparticles are separated by a small, but finite distance, the incident EM field polarises the nanoparticles. Figure 2.9 shows finite element method (FEM) simulation results of formation of hot spots. These plasmon fields interact with each other and induce opposite charges in the neighbouring particles, thus exposing analyte molecules residing in these gaps to strongly enhanced fields [40, 67]. The behaviour of these hot spots, which has been modelled using plane wave excitation can be found elsewhere [11, 68-70].

As a result of the enormous progress in nanotechnology, methods for fabricating SERS active substrates have attracted increasing interest. These kinds of substrates have several Page | 21

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advantages over the NPs in a solution, such as being easy to handle and locally concentrating the target species by adsorption on the surface. To create robust and reproducible SERS substrates, researchers have used a number of top-down, bottom-up and combined technologies for the last 40 years. Figure 2.10 shows SEM images of some of the SERS substrates fabricated during the time frame of 2009-2013 as reported by Luo et al. [10].

Top-down fabrication methods involve direct fabrication of small structures on a large area. Electron beam lithography (EBL) [1], focused ion beam (FIB) [71], metal-induced chemical etching [72] and sputtering [66, 73] are techniques commonly used by many research groups. The advantages of these include the ability to control size, shape and inter-particle distances of patterned structures. The main disadvantages are high production cost, complexity in fabrication and low throughput [10]. During the process of bottom-up fabrication, the larger systems are created from smaller units, including atoms, molecules, polymers and nanoparticles. Chemical synthesis, self-assembly, laser trapping and colloidal aggregation are some popular methods for preparing SERS substrates [4].

Figure 2.10: SEM images of various types of SERS active substrates fabricated by different techniques. Adapted from [10].

For the past few years, many diverse shapes have been fabricated, including spheres, cubes, bipyramids, nanorods, wires and nanoshells. Prediction of the locations of hot spots is not an easy task due to the low reproducibility. A commonly used direct method is the immobilisation of NPs on a substrate to create a SERS-active assembly. An intermediate layer of organic molecules such as aminopropyltriethoxysilane (APTES) and aminopropyl Page | 22

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trimethoxysilane (APTMS) is used for the immobilisation process due to their electrostatic or chemical interactions with the nanoparticles [2, 74]. Oblique angle deposition (OAD) is one of the most successful bottom-up techniques due to its relatively low cost and ease of fabrication [12, 73, 75, 76]. For the experimental work included in this thesis, OAD was used as the fabrication technique as the reproducibility is relatively good at a low cost. A detailed discussion of the OAD technique is presented in Section 3.1.2.

Moreover, researchers have further developed the fabrication processes by combining both top-down and bottom-up techniques. Some of the combined techniques found in the literature are metal cluster arrays combining EBL and immobilisation of NPs, localised oblique angle deposition using standard photolithography and silicon dry etching followed by vapour deposition at normal incidence [4, 10]. Template-assisted fabrication is another emerging technique to create various nanostructures. Anodised aluminium oxide (AAO) and Ag films over nanosphere (AgFON) substrates are used as SERS active substrates [77, 78]. The main advantage of the above techniques is the large average enhancement factor that arises due to well-controlled geometry of the nanostructures. The distribution of the EF is generally very broad on SERS substrates as molecules in hot spots usually dominate the average EF.

However, it is an ongoing challenge to achieve highly sensitive and reproducible SERS substrates. Depending on the specified application, researchers need to select their ideal SERS substrate. A uniform and reproducible substrate is essential for quantitative analysis, whereas a maximised SERS signal is required for trace analysis. Therefore, fabricating a SERS nanostructured substrate with a high density of hot spots is the key to a high EF [10]. A review article by Ko et al. [67] highlighted that SERS active systems must have features in the range of 5-100 nm. However, the higher SERS signals are generally observed for the nanostructures with a size of 20-70 nm. Nanoparticles that have the dimensions below 5 nm are starting to exhibit quantum effects as the small particles are deviating from the pseudo bulk plasmon behaviour leading to poor polarisability of the particle [67].

2.3.3 Applications of SERS

Applications of SERS have dramatically increased over the decades in the fields of material science, biochemistry and biosensing and even for single molecule detection [6, 7, 49, 79]. Table 2.3 shows a summary of some of the applications of SERS in different fields published by several group of researchers in no particular order. Reproducible and robust structures are most suitable for SERS. Performing a SERS experiment requires careful preparation of Page | 23

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the sample and an efficient optical set up to measure the maximum SERS signal. It is important to understand the limitations of SERS that constrain its broader application in the above-mentioned fields. The analyte must be located in very close proximity to the SERS substrate in order to be enhanced by the SERS phenomenon. If it fails to adsorb onto or remain close to SERS substrate, the possibility of SERS occurrence is very low. The orientation of the analyte with respect to the substrate is also important as it determines which vibrational modes are enhanced. In addition, the reproducibility of SERS enhancement is a critical factor for reliable characterisation and large-scale fabrication [80, 81].

Application Types of SERS substrates References

Detection of biomolecules - insulin analogs and antibody– Vacuum evaporated Ag films on [82] antigen binding glass - amino acid, phenylalanine and Vacuum deposited Ag on porous [83] antifreeze glycoprotein glass-ceramics Detection of explosives - 2,4-dinitrotoluene (2,4-DNT), Au coated femtosecond-laser- [84] 2,4-dinitrochlorobenzene (2,4- nanostructured sapphire DNCB), p-Nitroaniline, Nitrobenzene Multi-phase trace analyte detection Self-assembled Au and Ag colloids [42], [7] such as the aqueous, organic or air phases Detection of the influenza A virus Focused-ion-beam-fabricated [71] strain Au/Ag multilayered nanorod array Forensic science - detection of inks, finger-marks, Ag colloids, Ag-doped agarose gel [6] fire accelerants, gunshot disks, nanostructured Au by pulsed- residues etc. laser Detection of chemical and biological Au and Ag colloids [41], [79] molecules Detection of arsenic in water Ag sols and Ag nanoporous films [85] Detection of infectious agents and Vacuum deposited Ag nanorod array [86], [87] high sensitivity virus biosensing and classification Optical probing in biomedicine and Ag and Au nanoaggregates [88], [45] biophysics

Table 2.3: A brief summary of previously reported applications of SERS in the fields of chemical and biomolecular sensing.

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2.4 Geometrically-enhanced SERS

Generally, analyte detection has always been performed through the direct excitation of the SERS substrate. However, there have been some reports in the literature for “geometrically- enhanced” SERS signals due to the indirect excitation of the sample through a transparent substrate. For convenience and consistency in presenting the discussion, the terms of “near- side excitation” and “far-side excitation” will be used here, despite the different terminologies used by each group. Further, a schematic of the two illumination geometries is shown in Figure 2.11. In most of these experimental results, 180° backscattered SERS signals are collected for analysis, unless stated otherwise. a) Near-side excitation: The SERS substrate is directly excited through the air, i.e. the laser beam is directly focused onto the metal film with adsorbed analytes. b) Far-side excitation: This is the reverse geometry to the near-side. In this excitation set up, the laser light first penetrates the transparent substrate and then reaches the metal film and analytes. This situation is relevant to measurements of chemical processes (gas and liquid phase) performed through a SERS-active window or optical fibre probe.

Figure 2.11: Schematic of the near-side and far-side illumination of the SERS substrate.

This section is used to describe the literature of the geometrically-enhanced SERS signal with nanoscale rough metallic structures including discontinuous thin films. In 1984, Jennings et al. [14] used vacuum-deposited metal island films of silver and indium on glass slides with phthalocyanine (Pc) molecules as the analyte for SERS measurement at 514 nm excitation. The scattered signal was collected at 90° geometry. When the laser beam is focused directly onto the metal island film, the collected SERS signal is less than through the glass substrate. The 7.5 nm Pc film was formed by more than 12 monolayers in the stacking

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direction. They postulated that the extra enhancement comes from the far-side excitation of the Pc film is mainly due to the scattering arising from the first monolayers of Pc on the metal islands. However, they did not quantify this extra enhancement [14].

Nevertheless, this explanation was abandoned a few months later when Aroca et al. [15] observed SERS from a true monolayer. They revealed that electromagnetic enhancement is the predominant factor in SERS rather than the enhancement due to chemisorption. A few years later in 1989, Bello et al. [16] studied silver-coated titanium oxide (TiO2) particles on glass slides as SERS substrates. The TiO2 particles with a diameter of 0.2 µm provided the necessary surface roughness for the SERS process. The substrates were inserted in a cuvette of an ethanolic solution of analyte for the SERS measurement. The results showed an 11 increase in signal in far-side excitation geometry compared to near-side excitation. Even though this was consistent with the previously reported results, they suggested that further studies were needed to understand this excitation geometry dependence, where molecules forming the first layer may be only one contributing factor. Further they postulated, Raman radiation could be transmitted through the Ag layer as the laser beam was able to penetrate the continuous metal layer [16].

In the same year, Vo-Dinh et al. [17] used silver-coated microsphere glass slides as SERS substrates. They reported two times greater enhancement for far-side excitation when they measured the SERS intensity in a solution. The next experimental evidence is found back in 2000, when Viets and Hill [18] used vacuum-deposited Ag-island films on glass slides for SERS sensing with thiophenol molecules. They presented 2.2 higher SERS intensity for the far-side excitation than near-side excitation. In addition, even higher far-side enhancement was reported for a SERS substrate on the angle-polished tip of an optical fibre probe [18]. However, the origin of this additional enhancement has never been satisfactorily explained until recently.

In 2013, Funke and Wackerbarth [19] used evaporation to deposit silver and gold with a thickness of 40 nm on quartz glass slides and then studied the effect of the carrier material on SERS substrates. The nanostructures were assumed to be flattened spheres with a mean size of 45 nm and 60 nm for Ag and Au, respectively. A self-assembled monolayer of 4- nitrothiophenol was used as the analyte to measure the SERS signal from near-side and far- side illumination with an excitation wavelength of 785 nm. Interestingly, for the far-side SERS signal, the results show that the SERS signal is increased by 1.4 for Ag and 2.5 for Au compared to near-side SERS signal. The size-dependent dielectric function of a metallic NP, as described by the Drude model with the bulk optical constants for Ag and Au published

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by Johnson and Christy, was used to calculate the enhancement factor. Their theoretical model based on electromagnetic enhancement showed an enhancement factor of 1.7 for Ag and 5 for Au. They concluded that the increased SERS signal was originating from the more effective excitation of the localised surface plasmons when light passed from the quartz substrate. However, the theoretical calculation was purely based on a single metallic particle and the effects that particle aggregation and the distribution of particle size and shape might have on the local environment was not considered [19]. Their theoretical model is presented in Section 5.3.1.

Later in 2013, Jayawardhana et al. [20] confirmed the geometrically-enhanced SERS signal using Ag SERS substrates fabricated by thermal evaporation at an oblique angle of 86° on glass cover slips. For the SERS measurements, nanostructured films were functionalised with thiophenol and the signal was collected through the same objective in a backscattering geometry. The experimental results have shown up to 3 signal enhancement in far-side excitation geometry compared to the near-side. They concluded that this extra enhancement comes from the enhancement of the electric field at the boundary based on a simple analytical model derived from the Fresnel equations. The results were further supported by FDTD modelling representing the two excitation geometries but with relatively weak quantitative agreement with experiment [20]. The proposed Fresnel model is discussed further in Section 5.3.2.

In contrast to the fabrication techniques of SERS substrates discussed above, in 2015 Terekhov et al. [21] showed an increased SERS signal due to the far-side illumination of Ag nanoarrays fabricated on the surface of anodic aluminium oxide (AAO) substrates with CuTMPyP4 as the probing molecule and an excitation wavelength of 441.6 nm. The thickness of Ag coating has been varied to study how the spectral distance between the excitation wavelength and the extinction maximum influences the near-side and far-side SERS intensities. The increasing thickness of the Ag layer appears to impede the excitation radiation reaching the probing molecule in the far-side excitation geometry. The enhancement factors for Ag film thicknesses increasing from 15 nm, 30 nm, 45 nm and 60 nm are 1.55, 2.18, 1.57 and 1.15 respectively. Moreover, the results indicate that the far-side collected SERS signal is limited to Ag film thicknesses less than 60 nm. No discussion was presented to confirm or disclaim the previous theories for the origin of this far-side geometrically-enhanced SERS signal [21].

Other types of geometric enhancement have also been reported for SERS and Raman scattering. Surface plasmon resonance (SPR) spectroscopy is a well-known technique that

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has been successfully used for real-time quantification of proteins, DNA-drug interactions and self- assembled monolayers and it has been applied to clinical immunogenicity studies [57, 89]. The underlying principle of this non-invasive technique is the excitation of surface plasmon polaritons on the metal–air interface of planar substrates through attenuated total reflection (ATR). The two general experimental geometries used are Kretschmann-Raether (K-R) configuration and Otto configuration [55] as shown in Figure 2.12. In the first case, a thin metal layer is directly evaporated on top of a glass prism whereas for the latter set up, the prism is separated by a thin air gap from the metal film. The incident angle is kept greater than the critical angle of the glass-metal and glass-air interfaces for the K-R and Otto configurations, respectively. The evanescent field generated at the glass-metal interface decays through the metal layer and excites the SPP mode at the metal-air interface in the K- R configuration and the evanescent field generated at the glass-air interface is coupled to the SPP mode at the air-metal interface in the Otto arrangement.

Figure 2.12: Schematic of the incident beam path and the possible excitation of SPP at the metal-air interface for a) Kretschmann-Raether and b) Otto configurations.

The experimental work carried out by Futamata [90] showed that the Otto configuration improved the surface plasmon polariton-enhanced Raman scattering based on attenuated total reflection, thus increasing the sensitivity to analytes adsorbed onto Ag films. Coupling

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of the SPP for prism/air gap/SAM/silver was done with additional care to control the air gap size between the prism and the metal layer, which can typically be a few hundred nanometres.

Compared to the Otto configuration, the Kretschmann configuration seems to be the more favourable method when it is simultaneously combined with SPR for SERS sensing [91]. Even though planar thin films are reported to be poor SERS substrates in the absence of the surface roughness, Meyer et al. used a planar thin gold film of 50 nm to excite the SPP uniformly over a large area, thus overcoming the highly confined localised plasmon field in a narrow region of nanostructures using the Kretschmann configuration [92]. However, due to the angular dependence of the coupling of the SPP, the experimental set up required for the excitation of the substrate and collection of signal is quite complex compared to the standard backscatter SERS experiment. The most important additions to a SPP-coupled SERS sensing setup are a highly-collimated beam for the excitation and a high NA lens for improved collection efficiency.

Interestingly, Huo et al. reported a 30 enhancement in the Raman signal when it is collected in the reverse Kretschmann configuration compared to standard Kretschmann configuration [93]. This appears to be a comparison between near-side and far-side SPR- coupled Raman sensing, which has originally been described as the surface-plasmon- coupled directionally-enhanced Raman scattering. In the fabrication procedure, a 45 nm thick Ag layer with a 2 nm Cr adhesion layer was firstly deposited on a quartz glass slide. Then the sample was coated with 4-aminothiophenol to form the self-assembled monolayer. Finally, the sample was drop coated with colloidal Ag nanoparticles and attached to the prism using an index matching fluid for the measurements of near-side SERS and far-side SPP-enhanced Raman. The origin of the signal enhancement was described as the improvement of the collection efficiency of the Raman together with increasing coupling efficiency of localised plasmons of Ag NPs with propagating plasmons supported at the interface.

Whilst the above sources successfully report the geometrically-enhanced SERS signal, it can be further amplified by combining other geometric effects, including thin film interference [94-97], Fabry–Pérot cavities [98], far-field dipole coupling [99, 100] and whispering gallery modes [101-103] that are normally known as to generate enhanced electric fields.

Over the years, the far-side excitation geometry has only been used as a characterisation tool by most researchers. A complete understanding of the far-side geometrical enhancement in SERS, especially with the excitation of LSPR, has not yet been attained. The Page | 29

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reasons for the discrepancy between the experimental and theoretical approaches of the previously reported results are a major focus of this thesis, especially the future work recommended by Jayawardhana et al. [20] including investigation of the dependence of high refractive index substrates and angles of incidence. Therefore, in summary, this thesis is focusing on far-side geometrical enhancement because of concerns about the relatively poor qualitative agreement between experiment and theory, and the potential to combine with other geometrical enhancements such as thin film enhancements to boost the performance of SERS measurements through process windows and optical fibre probes.

2.5 Dielectric function of metal

Metals can be described as a plasma of electrons surrounding an immobile lattice of nuclei [67]. Close packing of atoms in bulk metals leads to a broadening of discrete energy levels of the free atom into bands due to the overlap of outer atomic orbitals. The outermost atomic orbital electrons are occupied in the conduction band and interact weakly with the nucleus. Consequently, these electrons mainly contribute to the electrical and thermal properties of the metals. The Fermi level is the highest occupied energy level in the conduction band, which is not completely filled in metals. The lower atomic orbitals (e.g.: nd, where n=3,4,5, for the noble metals Cu, Ag and Au, respectively) are the origin of the valence bands [104].

In the event of metal interaction with light, electrons are excited from an occupied state to an unoccupied state due to the absorption of energy. In most of the cases, the occupied state is either the valence band or the conduction band and the unoccupied state is obviously the incompletely filled conduction band. These electron transitions from the conduction band called as intraband transitions and the electron transitions from the valence band to the conduction band referred to as interband transitions. The latter transitions happen in the near UV-visible range and Figure 2.13 shows the schematic of the two transitions.

Bulk silver has five fully occupied d bands under Fermi level, a partially filled s band crossing the Fermi level and three empty p bands above the Fermi level [60]. Therefore, transitions within the s band are called intraband transitions and transitions from the d bands to s or p bands and transitions from the s band to p bands are called interband transitions. The energy threshold for interband transition of Ag and Au are ~4 eV and ~1.9 eV, respectively [59].

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Figure 2.13: Schematic diagram of the transition from discrete electronic levels in isolated atoms to electronic bands in the solid for noble metals. Adapted from [104].

The optical response of a metal can be described by the dielectric function and has contributions arising from intraband and interband electron transitions [59]. The dielectric function of a metal can be represented as a complex number and has a strong dependence on the wavelength or the frequency.

   intra     inter   . (2.15)

Conduction electrons in the noble metals of Ag and Au are primarily unbound and their contribution can be presented by the classical Drude model [105, 106] with the so called Drude-dielectric function:

2 2 2 p p p  b   11     i , (2.16) 2   2  2 22 i bb   b 

where  p is the plasma frequency and  b is the damping factor/collision frequency.

Generally, for metals the plasma frequency is in the UV-Vis range of 5-15 eV. However, the optical properties of metallic NPs and their thin films differ drastically from those of the bulk metal due to the confinement of conduction electrons, induced polarisation current confinement, cross-coupling between individual NPs and quantisation effects [60, 107]. Simulations based on density functional theory (DFT) have shown that as Ag NP size increases (larger than 10-20 nm), the intraband transitions shift to lower energies

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approaching the characteristics of bulk Ag [60]. The damping factor b 1 , where  is the relaxation time of free electrons that is normally on order of 10-14 s [55]. For small metallic NPs the damping factor is modified (Equation (2.17)) due to the significant increase in collisions between the electrons and the nanoparticle surface [60], leading to a reduced mean free path for the electrons [104].

A a   F , (2.17) b a

The symbol F is the Fermi velocity, A is a constant related to the scattering process and a is the radius of NP. Therefore, the size-dependent dielectric function for NPs as proposed by Noguez [108] can be written as shown in Equation (2.18) considering the contributions of free electrons, bound electrons and surface damping. The dielectric constant of the bulk metal bulk   is used as an initial approximation to calculate the contribution of interband transitions.

 ,,aa         NP  intra  bulk   intra    22   11  pp       22   bulk      i a    i b 

22  pp  . bulk   22 (2.18) i b  i   a 

2.6 Effective optical constants of metallic nanostructured films

Within the scope of this thesis, to theoretically model the geometrically-enhanced SERS signal, the absorbing nanostructured thin Ag film fabricated by oblique angle deposition needs to be represented by its effective optical constants. Ellipsometry is known to be a polarisation-dependent non-destructive technique that uses the change in the polarisation of the light to describe the optical properties of thin films, multilayer surfaces and composite nanostructured materials [59, 107, 109-113]. Ellipsometry modelling uses effective medium theory to represent optical properties of nanostructures thus incorporating their orientation and shape distribution. Therefore, ellipsometrically determined effective

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optical constants as represented by complex dielectric constants can be used to describe SERS enhancement by arrays of noble metal nanoparticles [114-117].

The complex dielectric function   and the complex refractive index n  of an absorbing material can be represented by

2 2     12 i   n     n  ik  , (2.19) where

 nk22 1 (2.20) 2  2nk, and, 1 and  2 are the real and imaginary parts of the dielectric function, and n and k are the refractive index and extinction coefficient, respectively.

Much work has been carried out to determine the effective optical constants of nanostructures including discontinuous thin films [59, 118, 119] and nanorods [120, 121] based on standard/spectroscopic ellipsometry and generalised ellipsometry [122]. The theories of standard ellipsometry and generalised ellipsometry will be discussed in Chapter 4.

Most of the previous ellipsometric analyses of thin Ag plasmonic films have assumed that the samples are isotropic or have dealt with relatively thick columnar structures. Maaroof et al. reported the effective optical constants of electrically percolated types of nanostructured thin silver films having a thickness less than 10 nm and the impact of an insulator coating using standard ellipsometry [123]. Even though their films were electrically continuous, nanoscale grain and void structures made a significant difference to the optical constants of bulk Ag as shown in Figure 2.14.

A study by Masten et al. reported ellipsometric studies on silver thin films epitaxially grown on silicon substrates and modelled the ellipsometric data with a two layered model [124]. Oates et al. studied the effective dielectric function of silver arrays deposited by evaporation onto patterned and unpatterned substrates [125]. The results have shown that random Ag NP arrays exhibit a uniaxial anisotropy, while ordered Ag NP arrays show a biaxial optical response. The corresponding effective optical constants for random arrays are depicted in Figure 2.15. The in-plane [ xy, ] dielectric function was modelled with a simple Lorentz oscillator and the out-of-plane [ z ] dielectric function was approximated by a

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Drude- Tauc- Lorentz model as similar to bulk data. The bulk silver optical constants were extracted from [126].

Figure 2.14: Derived effective optical constants of different metal layers deposited on glass a) refractive index and b) the extinction coefficient. Adapted from [123].

As can be seen in Figure 2.15, the in-plane dielectric function shows the LSPR centred on 2.7 eV and interband transitions at 3.8 eV, while the out-of-plane dielectric function is increased to account for surface scattering with a broadening of the Drude component compared to the bulk Ag.

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Ranjan et al. used generalised ellipsometry to derive the dielectric function of self-aligned silver nanoparticles on silicon ripple substrates [127]. SEM images of the samples showed that the structures grew preferentially along the ripples of the substrate and revealed biaxial anisotropy during the ellipsometric optical constant modelling. Further, they discussed in detail the significance of the strong coupling between nanostructures depending on the inter-particle gaps. In another study, Schmidt et al. used Mueller matrix ellipsometry to investigate the monoclinic optical constants, birefringence and dichroism of titanium nanocolumns deposited by glancing angle deposition [107]. These columnar thin films showed substantially different optical properties to those of the bulk material, including the absence of the characteristic interband transitions in the spectral range of 500-1000 nm.

Lončarić et al. reported the effective optical constants of Au island films deposited by e- beam evaporation [128]. The dispersion of the optical constants of films deposited with different thicknesses and temperatures have been discussed by applying different oscillators. These oscillators were used to represent different optical phenomena such as the LSPR, interband transitions and bulk plasmon resonance in the investigated spectral range. The ellipsometry modelling results showed that Gaussian oscillators provide better and more flexible fits for experimental data than the Lorentzian oscillators [128, 129]. With Page | 35

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the increasing thickness of gold island films, the imaginary part of the dielectric function, hence the LSPR, appeared to be red-shifted, together with an increase in the broadening and amplitude. However, increasing the substrate temperature results in a blue-shift in the LSPR position with a significant narrowing of the peak [128].

All of these studies confirm significant differences between the optical properties of bulk and nanoscale materials. Therefore, it is important to characterise the nanostructured Ag films used in this study as oblique angle deposited films are known to exhibit anisotropy for thicker coatings where tilted nanorod structures develop [107, 130]. The minimum thickness at which the anisotropy first appears and the point at which the bulk Ag properties develop is not yet known. Interestingly, according to a recent discussion about the reliability of the most widely used optical constants of Ag as published by Palik, and Johnson and Christy the data seemed to have some inconsistencies that could greatly affect the accuracy of many theoretical modelling and analysis [131].

2.7 Summary

In this Chapter, an overview of Raman scattering and surface-enhanced Raman scattering was presented, including substrate fabrication methods, their applications and the experimental evidence for geometrically-enhanced SERS. Then the use of ellipsometry to derive the effective optical constants of nanostructured thin metallic films was discussed. SERS has demonstrated its potential applications especially in the fields of bioscience, forensic and surface sciences over the past years with the rapid development of advanced nanofabrication techniques that can generate highly confined plasmonic fields around nanoscale metallic structures. While most of SERS community is focused on achieving high enhancement factors with high reproducibility and single molecular detection as discussed earlier in this Chapter, this thesis aims to understand the fundamentals of the geometrically- enhanced SERS signal, which has often been observed, but has not received any significant attention for the last 30 years. In particular, an improved understanding could provide a means to enhance the SERS signal when integrated with optical fibres for remote sensing. The far-side geometrical enhancement could be further developed to achieve more significant enhancement with the combination of other near-field enhancement methods, as presented in the literature review. The next Chapter discusses the experimental methods that will be used in this thesis.

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3 Experimental Methods

3.1 Sample fabrication

For the experimental work included in this thesis, oblique angle deposition (OAD) was used to fabricate nanostructured Ag thin films. Table 3.1 shows the key materials that were used for the fabrication process.

3.1.1 Materials

Materials Supplier Specifications Microscope slides Universal premium Plain unfrosted 7101-BU Pathology grade Thickness: 1-1.2 mm Size: 76.2  25.4 mm Sapphire substrates Cryscore Optoelectronic C-plane (0001) Double side polished Limited sapphire substrate Thickness: 500 μm Diameter: 100 mm Silver Sigma-Aldrich Co. LLC 327050 Aldrich Silver shot Particle size: 1-3 mm Assay: 99.99% trace metals basis Temperature RS Components Pty Ltd 555-392 sensitive labels Temperature range: 37-65 °C Film thickness ProSciTech Pty Ltd KC5464 monitor crystals

Table 3.1: List of materials used for the sample fabrication and analysis in this Chapter.

3.1.2 Oblique angle deposition (OAD)

Physical vapour deposition methods such as sputtering and thermal evaporation are commonly used techniques for thin-film fabrication [2, 10, 66]. Vacuum deposition is known to be a cost-effective technique to prepare SERS substrates. Discontinuous nanostructured films can be fabricated by thermal deposition at oblique angles by controlling the deposition rate of the metal during the evaporation process inside the vacuum chamber [31, 132]. OAD Page | 37

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has been demonstrated as a powerful technique for various plasmonic applications due to its ability to control shape, size and composition of nanostructures [76]. The geometric shadowing effect due to substrate tilting and low adatom mobility are the governing factors in the formation process of nanostructures with high porosity [73]. This method requires the vapour flux to reach the substrate at an oblique incidence of θ 70° as shown in Figure 3.1.

Figure 3.1: Oblique angle deposition and formation of nanoislands. Adapted from [133].

The steps associated with the formation of nanostructures during the deposition process is summarised below.

In the initial stage of the deposition, the vaporised metal atoms condense randomly onto the substrate. As the deposition proceeds, metal islands tend to grow and the shorter metal islands are shadowed by the taller islands. As a result of the substrate tilting, the condensed metal islands act as a barrier to the incoming vapour flux, thus preventing further growth in the shadowed regions. Consequently, the taller islands capture more vapour atoms and form a layer of nanocolumns tilted towards the incident vapour flux [73]. By adjusting the deposition parameters, one can form different morphologies of nanostructures as reported elsewhere [76], depending on the requirements of the practical application. However, there are large errors in monitoring the thickness and deposition rate during the deposition, as the sensors used in these systems cannot account for the tilting angle and the porosity of the film [75, 134].

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3.1.3 Emitech K975X turbo evaporator

The K975X is a vacuum coating unit that consists of an automatic vacuum pumping system, 22.8 cm diameter glass chamber, aluminium support collar and access ports (Figure 3.2). The pump down process is automatically controlled by a system microprocessor and the vacuum pressure is displayed digitally. The rotary stage is attached to a sliding access port and located at the side of the chamber. The stage can be titled from 0° to ± 180° with a precision of 0.1° by using the vernier scale attached externally. The coating unit can be used for two configuration modes of evaporation and sputtering [135]. In this work, the evaporation mode was used exclusively for the deposition of Ag whereas the sputtering mode was used to create an adhesion layer of Cr. A tungsten filament basket was used for metal evaporation with the metal of interest enclosed in the basket. An insulated shutter of 5 cm diameter was used to protect the sample from radiant heating and unwanted coating.

Figure 3.2: Photograph of the Emitech K975X turbo evaporator with parts labelled.

The thickness of the film was monitored by a quartz crystal film thickness monitor located inside the coating chamber. Generally, film thickness monitors (FTM) are used to determine or control the thickness of a material deposited under vacuum conditions. The system is working as an oscillator while one side of the crystal is exposed to the deposition source and as the deposition continues, material is gradually deposited on the crystal and increases

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the mass of the material deposited. As a result of this, the oscillation frequency of the crystal changes and indicates the thickness of the material deposited on the digital display. The method of mass determination based on the oscillation frequency of a quartz crystal was developed by G. Sauerbrey in 1959 [136]. The frequency change ( f ) of the oscillation is directly proportional to the added mass ( m ) and the corresponding relationship is expressed by the Sauerbrey equation:

2 f 2 fm   , (3.1) AGq q q

where f is the resonance frequency of the quartz crystal, Aq is the surface area of the quartz crystal and, q and Gq are the density and the shear modulus of the quartz, respectively.

During the initial stages of this work, a Cr target was used for sputtering and the glass microscope slides were placed on the rotary stage. Prior deposition of a thin Cr layer on glass is known to promote the adhesion of Ag [137]. It is important to note that this step was only used for the initial work on sample preparation (Section 6.3.1). Once the chamber was pumped down to a pressure of 710-3 mbar, the Ar bleed valve was opened. The sputter current set at 60 mA and the time to 90 s to achieve a thin Cr layer of 3 nm. Once a thickness of 3 nm was measured by the thickness monitor, the bleed valve was closed and the chamber was vented to atmosphere. Then the tungsten basket was filled with high purity Ag and operated at evaporation mode for the fabrication of nanostructured Ag films. Due to the different sample positioning requirements of different users, the distance from the evaporating source to the sample and to the thickness monitor was not kept the same for all coating runs. Therefore, it was important to apply a tooling factor (TF) to determine the actual thickness of the sample as shown in Figure 3.3.

The tooling factor is given by

2 d  sin  TF  ms    , (3.2) dsm  sin  where d s is the distance from the source to the stage, dm is the distance from the source to film thickness monitor, s is the angle between the plane of the stage and the source and

m is the angle between the plane of the thickness monitor and the source. Page | 40

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Figure 3.3: The sketch for sample positioning inside the vacuum chamber and the tooling factor calculation.

Therefore, the actual thickness ( t A ) of the sample can be found from ttAM TF , where tM is the thickness indicated on the digital display of the quartz crystal thickness monitor. The measured perpendicular distance (s =90°) from the source to the sample stage was 144 mm and the distance from source to the monitor was 165 mm. This lead to a tooling factor of 1.5. To avoid confusion when discussing different coating runs, the term “nominal thickness” is used to describe the thickness of the equivalent film deposited directly onto a sample surface in the perpendicular direction. The thickness of an OAD sample would be significantly smaller than this value due to the sample tilting.

3.1.3.1 Ag OAD films

Glass microscope slides were first cut into small sizes of 15  10 mm either using a diamond scribe (T961-90S, ProSciTech) or an automatic dicing saw (DAD 321) and ultrasonically cleaned with a sequence of acetone, ethanol and distilled water for 30 minutes and dried under a fume hood before thermal evaporation. The glass slides were placed in the Emitech K975X turbo evaporator attached to an aluminium L-channel using vacuum compatible carbon tape. The back side of the samples was covered with an aluminium foil to prevent the metal deposition on exposed surfaces. The sample tilting stage was used to tilt the sample with a precision of 0.1° during the oblique angle deposition process, as shown in Figure 3.4. The angle  was set as per experimental requirements and is described elsewhere. However, for most of the experiments discussed in this thesis, the angle was set to 86° to achieve the highest SERS signal [138].

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Figure 3.4: Schematic of the oblique angle deposition set up.

The samples were not rotated during coating. High purity silver (>99.99%, Sigma-Aldrich) was placed in the tungsten basket of the thermal evaporator. Once the chamber pumped down to a pressure of 110-5 mbar, a current of 8 A was applied to melt the Ag metal. The current was manually regulated to achieve a deposition rate of 0.03 nm s-1. A shutter was used to shield the sample during the initial heating of the metal until the evaporation rate was stable. The temperature inside the chamber was monitored using temperature sensitive labels (RS Components, Australia) and never rose above 37 °C. The thickness of the films was monitored using a thickness monitor (KC5464, ProSciTech, Australia) located inside the chamber near to the sample and mounted with surface normal parallel to the vapour flux. For some analysis, planar Ag samples were also deposited without any substrate tilting. The samples were immediately transferred to the desiccator after the deposition.

3.2 Sample imaging

3.2.1 Scanning electron microscopy (SEM)

Scanning electron microscopy uses a focused beam of high-energy electrons and provides sub-nanometre resolution for examining the structures of samples. During the electron- sample interaction, the high-energy electrons are dissipated through a variety of processes that produce secondary electrons, backscattered electrons, diffracted backscattered electrons and heat. Generally, the data collected from the electron-sample interaction over

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a selected area of the sample is due to the secondary and backscattered electrons that provide details of the morphology and topology of the sample. Sample preparation is minimal depending on the nature of the samples. This includes sample sizing to fit into the SEM chamber and methods to prevent charge build-up on electrically insulating samples. A Zeiss Supra 40VP SEM (Carl Zeiss SMT, Germany) was used to image samples at 3-5 kV for the work in this thesis. Most insulating samples are sputter coated before the imaging with a thin layer of conducting material such as carbon or gold [139]. No such coating was applied to maintain the electrical conductivity of the present samples, in order to avoid any changes in particle morphology. However, vacuum compatible carbon tapes were used to dissipate the charges on the surface of the sample due to the underlying dielectric substrate. Further, SEM images were analysed using ImageJ (Institutes of Health, USA) software to calculate the lengths and gap distances of the deposited Ag nanoparticles.

3.2.2 Atomic force microscopy (AFM)

Atomic force microscopy consists of an atomically sharp tip attached to a spring cantilever that is scanned across the sample surface at a selected rate [140]. The deflection of the cantilever caused by interactions between the sample and the tip is measured using an optical system with a laser. The most common imaging modes are the tapping mode and the contact mode. In the tapping mode, the tip is occasionally in contact with the sample whereas for the contact mode, it is constantly in contact with the sample during scanning. A Bruker Multimode 8 atomic force microscope was used for sample analysis with a RTESPA- 300 cantilever having a spring constant 40 N m-1. The tip radius was 8-12 nm. All images were processed with NanoScope Analysis 1.70 (Bruker, USA) software.

3.3 Sample characterisation

3.3.1 Optical profilometry

Optical profiling is a standard technique that is used to measure 3D surface topography and has been extended to measure the thickness of opaque and semitransparent films. This method stands as a non-destructive characterisation technique with rapid feedback. A 3D optical profilometer (Contour GT-K1, Bruker, Germany) was used for Ag film measurements with Vision 4.20 software (Bruker, Germany) for sample analysis. The thickness of the planar Ag and OAD films was determined by creating a step from the sample surface to the

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bare substrate. Depending on the feature height, phase shifting interferometry (PSI) and vertical scanning interferometry (VSI) modes were used to determine the film thickness. Generally, PSI is used to measure extremely smooth continuous surfaces such as mirrors and solar cell substrates, whereas VSI is used for relatively rough surfaces and discontinuous surfaces with steps.

3.3.2 Spectrophotometry

Spectrophotometry provides a quantitative measurement of transmission and reflection data of a sample of interest as a function of wavelength. The difference between the intensity of the incident light and the transmitted light is used to calculate the absorbance spectrum of the sample, as defined by the Beer-Lambert law. A Cary 50 UV-Vis spectrophotometer that uses a Xe flash lamp as the source of UV-Vis radiation was used for the sample measurements. Figure 3.5 shows schematic of the Cary 50 UV-Vis spectrophotometer set up.

Figure 3.5: Instrument set up of Cary 50 UV-Vis spectrophotometer. Adapted from [141].

The samples were loaded onto a customised holder located in the beam path before scanning. The Xe flash light is very intense and passes through a beam splitter to give simultaneous reference beam correction and only flashes when acquiring the data. This helps to eliminate excess photometric noise and the wavelength shift errors that are associated with traditional scanning methods. First, the light emitted by the Xe flash lamp Page | 44

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is collected by the Schwartzchild collector mirrors and then focused through a lens onto the excitation entry slit. Then the light is passed through an excitation monochromator, the sample and finally collected by a photomultiplier [141]. While the light is passing through the sample, some of the incident light will be absorbed by the sample, depending on its properties. The wavelength range of 300-1000 nm and a slow scan rate of 60 nm/min were selected for the present measurements.

3.3.3 Ellipsometry

Ellipsometry is a non-destructive optical technique that is used to investigate the dielectric properties of materials. A commercially available J. A. Woollam M-2000XI ellipsometer was used for the sample characterisation in this thesis. This ellipsometer is based on advanced diode array rotating compensator ellipsometry (DARCE) technology and uses a 75 W Xe lamp as the source [142]. The instrument is equipped with a polarisation state generator, an XY motorised stage, a polarisation state analyser, a detector and lamp power supply module and an electronics control module. The polarisation state generator consists of beam collimation optics, a fixed polariser and a compensator attached to a continuously rotating stepper motor. The polarisation analyser consists of a stepper motor driven polariser, four-quadrant detector, two beam-folding mirrors and two fibre coupling lenses at UV and IR spectral ranges. Fibre optic cables are used to couple the light from the polarisation analyser to the detector which is located in the detector and lamp supply module. The detector and lamp supply module contains UV and IR spectrometers which covers the wavelength range of 245-1690 nm and also houses the power supply of the lamp.

For the standard ellipsometry presented in this thesis, the samples were analysed with focusing probes attached to the system as shown in Figure 3.6. However, attaching a manual rotation stage on top of the ellipsometer stage limits the space available to accommodate the focusing probes during the Mueller matrix ellipsometry. CompleteEASE (5.11a, J. A. Woollam Co., Inc) software was used to control the instrument during the measurement and to extract the optical constants from a user-built optical model representing the nominal structure of the sample (Sections 4.5.1 and 4.7.1).

A system check was done using the 25 nm SiO2 calibration wafer supplied by the manufacturer prior to any analysis. The system-built optical model was then automatically fitted to the measurement of the calibration wafer. After loading new samples on the ellipsometer stage, each sample surface must be aligned with the ellipsometer optics. This procedure includes the complete alignment of sample tip/ tilt and sample height. The Page | 45

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samples were then analysed for at least three incidence angles ranging from 50°-70° with an acquisition time of 10 s in high accuracy mode.

Figure 3.6: Instrument set up of J. A. Woollam M-2000XI ellipsometer with focus probes.

For the analysis of anisotropic samples, Mueller matrix ellipsometry was performed using a manual rotation stage attached on top of the ellipsometer stage as indicated earlier. The instrument only has the capability of measuring the first three rows of the Mueller matrix and the measurements were recorded at 37 in-plane clockwise rotation angles, from 0° to 360° with respect to the direction of the deposition vapour flux. For an isotropic sample, Mueller matrix has only three independent parameters and in contrast an anisotropic sample can be expressed in six independent parameters. In the event of depolarisation effect in an anisotropic sample, at least seven parameters are needed [143].

3.3.4 Raman spectroscopy

Raman spectroscopy can be thought of as a finger print detection technique for molecules as discussed in Chapter 2. A Renishaw inVia Raman spectrometer coupled to a research grade Leica microscope was used for SERS measurements in this thesis. The instrument has five different wavelengths for excitation; argon-ion laser (457.9 nm, 488.0 nm and 514.5 nm), helium-neon laser (632.8 nm) and diode laser (785 nm). The main unit of the Renishaw Raman spectrometer consists of a microscope, a system unit and a Peltier-cooled charged-coupled device (CCD) as shown in Figure 3.7 a). The system unit is equipped with

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an optical arrangement that guides the laser light through to the microscope head and collects the backscattered signal as shown in Figure 3.7 b). The holographic notch filter rejects the Rayleigh scattered light and only transmits the Raman component onto the entrance slit.

Figure 3.7: Photographs of a) Renishaw inVia Raman spectroscope and b) a more detailed view of the optical setup used to guide light to the microscope and CCD.

The signal measurement can be done in two different configurations, namely static scanning and extended scanning. In the static scanning mode, the spectrometer grating is held stationary while the light is dispersed onto the CCD whereas in the extended mode the grating is rotated to collect a wide spectral range. The grating determines the resolution of the spectrum: the more lines/mm the higher the resolution. The slit width is 65 µm in standard confocal configuration and is 20 µm for high confocal mode. Matched

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combinations of grating and filter were installed to match the laser source before each experiment, as indicated in Table 3.2. Once the laser has been allowed to heat up and stabilise, the instrument is calibrated using both an internal Si reference and a manually measured Si Raman peak at 520 cm-1 prior to the sample analysis. These steps help to confirm the wavelength calibration as well as the throughput check. Samples are normally placed on a motorised XYZ stage that is fitted to the microscope and controlled by the WiRE 4.1 software. A colour video camera attached to the microscope was used for the laser alignment, positioning and focusing on the sample.

Wavelength (nm) 1200 l/mm 1800 l/mm 2400 l/mm 457  488  514 Streamline  633 Streamline  785 

The microscope is fitted out with five different objectives and their details are summarised in Table 3.3. The laser power was measured after each objective at each excitation wavelength using a Fieldmaster (Coherent) laser power meter for the work in this thesis. It is highly recommended by the manufacturer to use the high NA lenses (especially the 50 and 100) to collect spectra, as the instrument is optimised for high spatial resolution Raman spectral mapping.

Objective NA Solid angle Half angle WD 100% power (mW) DOF (sr) (θ) (mm) of 514 nm laser (μm) 5 0.12 0.045 6.9 14 11.1 17.8 20 0.4 0.525 23.6 1.15 10.9 1.6 50L 0.5 0.842 30.0 8.2 5.8 1 50 0.75 2.127 48.6 0.5 8.3 0.46 100 0.85 2.97 58.2 0.33 2.7 0.36

Table 3.3: List of objectives attached to the Renishaw inVia Raman microscope.

Ag OAD samples were attached to a customised holder to excite both near-side and far-side geometries during the analysis. Considering the near normal incidence of light onto the sample, the lowest NA lens (due to its low cone half angle) was used to excite samples and to collect the 180° backscattered Raman signal.

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The intrinsic fluorescence background of the SERS spectra was removed using the penalised least squares method reported by Cadusch et al. [144]. In this automated algorithm, a single parameter  (the stiffness parameter) is used to determine the derived fluorescence background of the Raman spectrum and can be varied from 0-2106. Once this single parameter is fixed to remove the background, it can be applied for a series of related spectra for a more consistent and reliable analysis. This method provides an advantage over manual polynomial fitting, as it requires no user intervention to select the background curve fitting points over a spectral range of the Raman spectrum. An example of the background removal process is shown in Figure 3.8 for a thiophenol SERS spectrum recorded at 633 nm excitation.

As mentioned earlier (Table 3.2), Streamline is a line scanning mapping technique that is used to create a Raman map over a large sample area. During the mapping, several Raman spectra of the sample are collected simultaneously. This technique shortens the long acquisition times linked to conventional point-by-point mapping by up to 20 times for millimetre-sized sample areas. A detailed discussion of the technique and some applications are found elsewhere [145, 146].

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3.3.5 X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy is a semi-quantitative technique that used to examine the chemical composition at the surface of a sample. As a surface-sensitive analytical technique, it provides useful comparative information to SERS. X-rays of specific energies are used to excite the atoms of the sample, leading to the ejection of photoelectrons. The energies of the photoelectrons are well defined in terms of the binding energies of their parent atoms and this leads to the peaks in the XPS spectrum. The peak intensity is proportional to the number of atoms within the outer 10 nm of the surface, whereas the peak position is an indication of the elemental and chemical composition [147]. For the analysis of transparent substrates and Ag OAD samples, the XPS spectra were collected using a Kratos Axis Nova instrument

(Kratos, Manchester, UK) with a monochromated Al Kα source (source energy 1486.69 eV) at a power of 150 W. To identify all of the elements presented on the sample, a survey spectrum was firstly collected at a pass energy of 160 eV. Then to obtain more detailed information about chemical bonding, high-resolution spectra were recorded from individual peaks at 20 eV pass energy. Further, the data quantification was performed with CasaXPS 2.3.15 software (Casa Software Ltd., UK) using the sensitivity factors provided by the manufacturer.

3.4 Summary

In this chapter, the materials that were used to fabricate nanostructured Ag thin films and the experimental methods that were used to investigate their geometrical, optical and plasmonic properties were discussed. Oblique angle deposition, a low-cost method was used for fabrication of reproducible nanostructured Ag thin films. The geometrical and structural properties of the Ag thin films were investigated using the techniques of profilometry and sample imaging by SEM and AFM. Spectrophotometry and ellipsometry were used to determine the effective optical response of the samples, whereas SERS analysis was performed using the Renishaw inVia Raman microscope. The chemical composition of the samples was also investigated by XPS.

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4 Effective Optical Constants of Nanostructured Ag Films

4.1 Introduction

Finite-difference time-domain (FDTD) modelling and other computational electrodynamic methods such as discrete-dipole approximation (DDA) and finite element method (FEM) are used to determine local electric fields by solving Maxwell’s equations. They generally use the bulk properties of the material to generate the field intensities around nanoscale plasmonic structures [20, 96, 148, 149]. While the local fields help to understand some aspects of these plasmonic films, such as ‘hot spots’, they do not provide a convenient description of the average macroscopic behaviour. In contrast, ellipsometric measurements provide a spatial average of thin films [112, 118, 150], polymer films [151], multilayer surfaces [110, 152, 153] and composite nanostructured materials [154, 155] on length scales that are relevant to geometrical optics. Use of ellipsometry serves an additional purpose of averaging the ellipsometric measurements over a probe area of a few hundred microns, thus incorporating information from a wide distribution of particle sizes. The key result is the dielectric constant, where the nanoscale displacements convert into a macroscopic average that represents the effective response of the nanoscale material to an incident electromagnetic field. This allows macroscopic phenomena such as wave transmission and reflection to be described through the Fresnel equations using the complex refractive index of a material that is directly related to the dielectric constant [143]. There have been many different theoretical approaches used to explain the near-field enhancement in SERS. Some commonly discussed methods include DDA calculations [116, 156], effective medium theories [115, 157], FDTD modelling [158-160] and Fresnel equations [20, 117, 161]. Therefore, this Chapter covers the ellipsometric study of the effective optical constants of Ag OAD films and most importantly the Ag OAD86° 100 nm film that will later be used to calculate the geometrically-enhanced SERS signal using the extended Fresnel model in Chapter 5.

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4.2 Determination of film thickness by profilometry

Firstly, Ag films with a nominal thickness of 100 nm were deposited on pre-cleaned glass slides without any substrate tilting. Half of the area on the glass slide was covered with a cover slip to create a sharp step between the coated and uncoated regions. The same procedure was followed for the deposition of Ag OAD86° 100 nm films together with the tilting of the sample stage in the coating unit to set the angle between the substrate normal and the vapour flux to be 86°. Prior to the profilometry, the coverslips that were used to create the step on the substrate surface were removed. Figure 4.1 shows typical zoomed in views of the steps measured on of Ag planar and OAD86° films.

a) Ag planar 100 nm

b) Ag OAD86° 100 nm

Figure 4.1: Examples of step slides created on a) planar Ag film and b) Ag OAD86° film with a nominal thickness of 100 nm.

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Two clean areas on both sides of the step were selected and the modal tilt removal was applied in data processing before calculating the average thickness of the samples. The areas immediately next to the step were avoided in order to remove any shadowing effects. The average sample thickness and the standard deviation as determined by the Vision 4.20 software were found to be 98 ± 13 nm and 15 ± 5 nm for Ag planar and OAD86° films of nominal thickness of 100 nm, respectively. This experiment was mainly performed as a check of the film thickness monitor reading of the Emitech coating unit and was not used to measure the thickness of any other OAD films discussed later in this thesis. It confirmed that the thickness monitor was correctly calibrated for the purposes of this work. Although the OAD film is known to consist of nanoscale islands [138], the optical profilometer cannot resolve these sub wavelength structure and is therefore assumed to measure the thickness of the film as an effective medium (Section 4.5.2).

4.3 Spectrophotometry of planar Ag films and Ag OAD films

Prior to the ellipsometry analysis to determine the effective optical constants of nanostructured Ag films, a set of samples including a neutral density filter, microscope glass slide, planar Ag films and Ag OAD films were used for transmission measurements in the Cary 50 UV-Vis spectrophotometer. The results are shown in Figure 4.2. The transmission measured through the neutral density filter (OD1) and microscope glass suggests that the accuracy of the measurements was acceptable with mean values around 10%T and 92%T for OD1 filter and glass, respectively. As can be seen clearly, the glass is highly absorbing for wavelengths less than 350 nm due to its band gap around 4 eV originating from the mixture of materials included in glass, mainly silicon dioxide and sodium oxide [162].

The Ag 100 nm film that was deposited at normal incidence to the deposition vapour flux shows the lowest light transmission in the measured wavelength range. It is interesting to note that the Ag OAD films especially OAD80° and OAD86° exhibit the characteristic LSPR behaviour with minimum transmission around 500 nm (Section 2.3.1). The weak dips around 725 nm were later identified to originate from the source polarisation of the Xe lamp. Noticeably, Ag 50 nm and OAD70° films have similar characteristics as thin continuous films with no LSPR. This thesis focused on the initial nanoisland growth stage of the deposition, as thin island films have significant optical transmission compared to the rod-like structures that form in thicker OAD films [163]. Although the coating thickness for Page | 53

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deposition in the normal direction was measured to be close to 100 nm, the true film thickness of the OAD samples is significantly less than 100 nm, as seen from the visual inspection and Figure 4.1.

Figure 4.2: The light transmission through OD1, microscope glass, planar Ag films and Ag OAD films measured with Cary 50 UV-Vis spectrophotometer. The Ag OAD films have a nominal thickness of 100 nm.

Noble metal nanoparticles have strong size-dependent optical properties, revealing a distinct UV-visible extinction band that is not present in the bulk metal spectrum and arises as a result of LSPR [54]. It has been shown that this LSPR in the absorption spectrum is strongly dependent upon the size, shape and inter-particle spacing of the nanoparticles, and the dielectric properties of the local environment including the substrate as discussed in Section 2.3.1 [43, 54, 164-166].

The absorbance ( A ) of the films was calculated using the relationship:

ATlog10  1   2  log10  %T . (4.1)

The UV-Vis spectra give additional information about the wavelength dependence of the extinction coefficient of the thin absorbing films, as shown in Figure 4.3. The relationship between absorbance ( ) and the extinction coefficient ( k ) can be expressed by

4 k A d d , (4.2) 

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where  is the absorption coefficient and d is the thickness of the film. When the medium is non-absorbing = k =0.

The absorbance minimum at 320 nm is characteristic of silver and occurs due to the lowest interband electron transitions [59]. LSPR maxima are clearly seen in the OAD80° and OAD86° films around 500 nm, suggesting plasmonic behaviour as suitable candidates for SERS sensing. The thicker OAD70° film is more characteristic of bulk silver.

4.4 Image analysis of Ag OAD films

SEM images of the Ag OAD films of 70°, 80° and 86° with a nominal thickness of 100 nm were processed using ImageJ software to determine the Area% of NPs, average particle count and average particle size. The results of the analysis are shown in Figure 4.4. The results suggest that as the oblique angle increase, the nanostructures tend to form island- like structures rather than the large clusters that can be seen in the Ag OAD70° film. Increasing the number of particles in a given area means an increased probability of hot spot formation for SERS sensing. Further, AFM images of the samples were taken to study the structure and surface roughness of the samples. The corresponding images are shown in Figure 4.5.

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a) Ag OAD 70°

% Area : 79.0 Count : 12 per μm2 Average size : 86400 nm2

b) Ag OAD80°

% Area : 60.6 Count : 412 per μm2 Average size : 2400 nm2

c) Ag OAD86°

% Area : 48.6 Count : 1428 per μm2 Average size : 700 nm2

Figure 4.4: SEM images (left) of Ag OAD films deposited at a) 70° , b) 80° and c) 86° (scale bar 100 nm) and the corresponding ImageJ analysis results (right).

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a) Ag OAD70°

b) Ag OAD80°

c) Ag OAD86°

Figure 4.5: 2D view (left) and 3D view (right) of the AFM images of samples of Ag a) OAD70°, b) OAD80° and c) OAD86°.

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As can be seen in the AFM images, the OAD70° film has larger structures aggregated with each other compared to the other two samples which are more island-like. A detailed image analysis of the AFM images was performed by the NanoScope Analysis 1.70 software. The results are shown by box plots in Figure 4.6 and the median, first quartile and third quartile values are indicated on the plots, while the error bars signify the maximum and minimum.

Figure 4.6: Box plots of the a) nanoparticle area and b) height calculated using the AFM images of OAD70°, OAD80° and OAD86° films by the NanoScope Analysis software.

The results generally agree with the SEM image analysis results with the OAD70° film having the largest particle area and height. However, the large relative errors in the AFM results prevented a detailed comparison with the SEM image analysis. This is probably due to the fact that the AFM probe cannot scan down to the substrate between closely spaced nanoparticles because of the pyramidal shape of the tip. Having established the general structural characteristics of the Ag planar and OAD films, from this point onwards, ellipsometry was used to determine the wavelength-dependent effective optical constants and the effective film thickness of the Ag planar and OAD films. Page | 58

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4.5 Standard ellipsometry

4.5.1 Theory

Ellipsometry is a non-destructive technique that measures the change in the polarisation state of the incident light reflected from a smooth surface. In general, ellipsometry measurements are performed in the UV and visible region while some have been reported including the infrared region depending on the material and the optical application [109, 167, 168]. Two main restrictions in ellipsometry measurements are, that the roughness of samples needs to be rather small and the data must be collected at oblique angles. In cases where light is scattered severely by the surface roughness of samples, the collected reflected light intensity becomes weaker. This will increase the errors in the ellipsometry measurements. It has been reported that the roughness of the structures should be less than 30% of the measurement wavelength to acquire a good measurement [169]. Ellipsometry has several advantages over other spectroscopic techniques in terms of high sensitivity and easy real-time monitoring. However, there remain some challenges for the data analysis, as this technique is an indirect measurement. Building an optical model to represent the measurements tends to be complicated, which requires knowledge, experience and patience.

The most widely used theoretical descriptions to represent polarised light are the Jones formalism and the Stokes-Mueller formalism [143]. Generally, totally polarised light is described by the Jones formalism, while an arbitrary polarisation can be represented by the Mueller-Stokes formalism. When a light ray propagates through an isotropic medium along the z direction, the electric field ( E ) is confined in the x-y plane which is perpendicular to z. For totally polarised light, this electric field can be described by an ellipse as shown in Figure 4.7. The electric field can be resolved along the x and y directions having a real amplitude ( A ) and phase ( ).

Ex A xexp i x , (4.3)

Ey A yexp i y . (4.4)

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Figure 4.7: Representation of elliptically polarised light.

The Jones vector describes the polarisation of light as a transverse wave. Augustin Jean Fresnel has showed that the polarisation of light waves was possible, if the light was in the form of a transverse wave rather than a longitudinal wave [143]. Therefore, in general representation of polarised light, an electromagnetic wave with orthogonal electric and magnetic fields is used as characteristic of light representing a transverse electromagnetic wave. In the absence of depolarisation, the Jones matrix ( J ) connects the Jones vector of the incident light to the Jones vector of the output light as defined below.

out  in  EEppJJpp ps     . (4.5) out  JJin  EEss  sp ss  

For planar and isotropic samples, the Jones matrix forms a simple diagonal matrix. The diagonal elements of the Jones matrix can be expressed in terms of Fresnel reflection coefficients that are defined for fields parallel ( rp ) and perpendicular ( rs ) to the plane of incidence.

out  in  Ep r p 0 Ep     . (4.6) out 0 r in  EEss s  

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In the simplest case of isotropic samples, standard ellipsometry (SE) measures the ellipsometric angles psi (  ) and delta (  ). These angles are used to describe the change in the polarisation of the light after interaction with the sample and are defined from the complex ratio  (Equation (4.7)).

rp   tan  expi   , (4.7) rs

rp where tan  and   ps   . The quantities tan and are known as the rs amplitude ratio and phase shift, respectively. As ellipsometry measures these two magnitudes at each wavelength, the information obtained from this technique is greater than simple reflection measurements. An ellipsometer is basically made of two optical arms with a sample holder in between. The first arm compromises a light source and a polarisation state generator (PSG) and the second arm consists of a polarisation state analyser (PSA) and a detector. An ellipsometer aligned with high quality optics is capable of measuring and to a precision of about 0.01-0.02° leading to a theoretical sensitivity to the film thickness on the order of 0.01 nm [122].

As ellipsometry is an indirect measurement, an optical model must be built to determine the optical constants of the sample. The model-based data analysis starts with a layered optical model which represents the nominal structure of the sample and is used to generate model data for comparison with the measurements. The optical properties thus determined can be either refractive index for a transparent substrate and the complex refractive index/complex dielectric function for an absorbing film. The quality of the fit between the model and the measured data is assessed by a figure of merit and the most popular method is based on the mean-squared error (MSE,SE ) of the differences between the measured and the model-generated data:

cc22 1 S          2 j j j j SE      , (4.8) 21SK     j1 jj    where denotes the number of measured data pairs ( , ), K is the number of real- S j j

cc valued model parameters, ( jj, ) are the model-generated parameters at E   j and

 (jj, ) are the standard deviations obtained during the measurements. For an ideal Page | 61

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sample, the MSE should be close to one, however, when dealing with real life samples having surface structures, MSE values as high as 10-20 may be considered acceptable given the complexity of the model [170]. Nevertheless, the accuracy of the optical model determines the physical meaningfulness of the derived optical constants.

The sensitivity of the ellipsometer measurements to the refractive index derived from optical model is reported within 0.01-0.0001 RIU [122,1 150]. However,, 17 it should be noted that the sensitivity of the refractive index often depends on limits of systematic errors in the ellipsometry measurements including calibrations errors associated with the angle of incidence and wavelength and azimuthal alignments of polarisation elements of the ellipsometer. In addition to the uncertainty introduced by these systematic errors that fundamentally limit the sensitivity of the optical constants, the uncertainty is also ellipsometer optical model-dependent. As the complexity of optical model increases, especially when it is combined with multiple layers, grading, surface roughness, back surface reflections and anisotropy, the uncertainty in optical constants will tend towards the higher end of the range [172].

4.5.2 Results

For standard ellipsometry measurements, psi (  ) and delta (  ) data were recorded at three different incidence angles of 55°, 60° and 65° for each sample. Bare glass and sapphire substrate refractive indices were modelled with a Cauchy dispersion model in CompleteEASE software [170] and the Ag planar and nanostructured films were modelled with a B-Spline layer starting from an unknown material. The Cauchy dispersion equation that represents the index of refraction of a transparent substrate [118, 173] is represented by Equation (4.9), where A gives the estimated amplitude of the index for the material, and B and C maintain the shape and curvature of the index against the wavelength.

BC nA     . (4.9) 24

To avoid backside surface reflections from transparent substrates, a piece of scotch tape was attached to the glass slide, while a single-side sandblasted sapphire substrate was used as required. The wavelength-dependent refractive index derived from the Cauchy model for glass and sapphire are shown in Figure 4.8 . The MSE of the model was less than 3.7 for glass and 5.1 for sapphire.

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A single layer B-Spline optical model in CompleteEASE software was used to describe the nominal structure of the Ag planar and OAD films. B-Spline is a basis set of polynomial splines that has control points equally spaced in the wavelength range or photon energy to determine the wavelength-dependent optical constants of a sample. A more detailed description of the B-Spline is found elsewhere [174]. The B-Spline model with Kramers- Kronig (KK) consistency mode ON helps to constrain the optical constants to follow a physical shape, thus assuring that the model-represented optical constants are physically valid [170]. Applying a KK consistent B-Spline offers an internal coupling between the real and imaginary parts of the dielectric function, where the real part is defined by the KK transform of the imaginary part [111]. The detailed equations that represent the KK transformation can be found elsewhere [143]. The wavelength or frequency-dependent complex refractive index and complex dielectric function are related by Equations (2.19) and (2.20). The model-generated n and k values for Ag planar films on glass substrates based on standard ellipsometry measurements are shown in Figure 4.9 together with the bulk silver values of Palik [126]. The corresponding effective thickness of Ag planar 100 nm and 50 nm films as determined by the B-Spline model were 83 ± 2 nm and 44 ± 4 nm respectively, when MSE was less than four.

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Figure 4.9: The effective optical constants of planar Ag 50 nm and 100 nm films as derived by the B-Spline model in CompleteEASE software and compared with the bulk silver values of Palik.

The effective optical constants of the planar Ag 100 nm thin film are in good agreement with the published optical constants for bulk silver [126, 131]. The extracted optical constants of the planar Ag 50 nm film do not agree with the Palik values, with the refractive index having a significant difference. The real part of the complex refractive index ( n ) is known to be highly sensitive to grain structure and crystal defects [175]. The SEM image of the planar Ag 50 nm film showed metal globes randomly distributed on the substrate. Therefore the film was clearly far from being a smooth continuous layer. It is also important to note that as shown by the work of Jiang et al. [131], there remain some significant differences between the most commonly used and referenced Ag optical constants due to the extreme sensitivity to sample preparation methods and exposure to ambient air. During exposure to laboratory conditions, the surface composition of the Ag substrates can be changed due to oxidation and surface contamination from adventitious carbon [176].

Following a similar procedure to the one described earlier, the effective optical constants of Ag OAD70°, OAD80° and OAD86° with a nominal thickness of 100 nm were extracted from the B-Spline layer model based on standard ellipsometry measurements with the parameters constrained to follow the KK consistency condition. As can be seen in Figure 4.10, compared to the OAD70° film, the OAD80° and OAD86° films show a very prominent plasmon resonance peak in the extinction coefficient around 500 nm. Similar patterns were observed for the absorption measurements of these films (Figure 4.3). Page | 64

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Figure 4.10: Effective optical constants of Ag OAD70°, OAD80° and OAD86° films on glass substrates as determined by the B-Spline optical model.

These optical constants were generated based on the simplest case of isotropic materials with no directional dependence, where the dielectric function becomes a scalar. A detailed explanation of the connection between the dielectric tensor and the intrinsic polarisabilities of a material is found elsewhere [143, 169]. From the results in Figure 4.10, it appears that the wavelength-dependent effective optical constants of thin nanostructured Ag films are highly dependent on the geometrical features of the films, including the particle size, shape and thickness. These variations can be clearly seen from the significant differences between these OAD films in the SEM and AFM images (Figure 4.4 and Figure 4.5). The B-Spline layer suggests that the effective thickness of the Ag OAD films of 70°, 80° and 86° was 44 ± 3 nm, 21 ± 2 nm and 15 ± 2 nm, respectively. The MSE values were less than eight for all three OAD films. Remarkably, the work by Tompkins et al. [177] showed that the effective optical constants of thin platinum metal films fabricated by evaporation and sputtering are not the same, suggesting that it is important to determine the optical constants of the specific material of interest, without directly using the handbook values for any data analysis.

The main focus of the ellipsometry analysis in this thesis is to model the effective optical constants of Ag OAD86° 100 nm SERS-active films having disconnected island-like structures as shown in Figure 4.4 c) and Figure 4.11. These films have been used to identify the extra SERS signal enhancement due to the far-side excitation geometry of the substrate because they are relatively repeatable and straightforward to fabricate [20]. A hemispherical shape with particle radius of 15-20 nm has been assumed for the FDTD Page | 65

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modelling reported by Jayawardhana et al. [20]. However, the absorption measurements performed at four different sample plane rotations revealed an anisotropic optical response of the Ag OAD86° nanostructures for light at normal incidence. Therefore some additional ellipsometry analysis was conducted to assess the anisotropy.

Figure 4.11: Cross-sectional SEM image of a Ag OAD86° 100 nm film. Vapour flux is from right to left during the oblique angle deposition.

4.6 Anisotropy of Ag OAD86°

Absorption measurements performed at four different sample plane rotations revealed an anisotropic optical response of the Ag OAD86° 100 nm nanostructures for light at normal incidence as shown in Figure 4.12. The Ag OAD86° 100 nm film was further used for four in-plane rotations during standard ellipsometry measurements in order to explore the observed anisotropy. The sample was rotated in 90° steps in the clockwise direction when viewed from above, as shown in the inset of Figure 4.13. If the layer is assumed to be homogeneous and isotropic, the model-generated results shown in Figure 4.13, indicate significant changes in n and k related to the direction of the vapour flux during deposition. This confirms the optical anisotropy for polarised measurements in directions parallel and perpendicular to the vapour flux. The model-generated thickness values shown in Table 4.1, also indicate that the B-Spline model is strongly sensitive to the rotation angle.

Rotation angle 0° 90° 180° 270° Effective thickness [nm] 13 ± 3 16  4 13  3 16  4

Table 4.1: Effective film thickness of the Ag OAD86° 100 nm film corresponding to the best match model for four sample plane rotation angles in 90° steps.

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Further, the measured lengths and the gap distance of nanoparticles in directions parallel and perpendicular to the vapour flux show a small asymmetry, which supports the observed anisotropy. The particle size distributions are shown by box plots in Figure 4.14 where the median, first quartile and third quartile values are indicated on the plots, while the error bars signify the maximum and minimum.

In thicker metallic films that have developed nanocolumns or rods, the structures are generally inclined from the substrate normal towards the deposition vapour flux [12, 163, 178]. Abelmann and Lodder [179] discuss the influence of the bundling effect and the geometric shadowing effect in oblique angle deposition to describe the size distribution of

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nanoparticles along two orthogonal directions in the early stages of the film growth. The structural growth starts as a random distribution of nucleation sites, which act as a location for the further growth of the film. As soon as the nucleus is formed, it casts a shadow parallel to the vapour flux. This will block the formation of a secondary nucleus, thus leaving an empty space behind. When the first nucleus grows, due to the conservation of parallel momentum, the adatoms also move in a direction perpendicular to the incident vapour, which induces a bundling of the nanostructures. However, the overall effect of conservation of momentum on the bundling effect is much smaller compared to the effect of self- shadowing as explained by the simulations in [179].

As discussed above, the sample-plane rotated standard ellipsometry measurements, the absorption measurements and even the SEM image analysis results confirm that the Ag OAD86° 100 nm nanostructures are anisotropic, even though the film thickness is less than 20 nm. Therefore, a refined optical model is needed to derive the effective optical constants of the Ag OAD86° 100 nm film and a suitable model is based on the work of Oates et al. [125] using variable angle Mueller matrix ellipsometry. This will help to accurately represent the effective optical response of the Ag OAD86° 100 nm film in order to calculate the geometrically-enhanced SERS signal based on the Fresnel equations using complex reflection and transmission coefficients.

4.7 Mueller matrix ellipsometry

4.7.1 Theory

In contrast to standard ellipsometry which only measures the diagonal elements of the Jones matrix for the analysis of isotropic samples, off-diagonal elements of the Jones matrix become non-zero for optically anisotropic samples that convert p polarised light into s polarised light and vice versa [120]. Therefore, generalised ellipsometry has been introduced as an extension of standard ellipsometry that includes both Jones matrix ellipsometry and Mueller matrix ellipsometry to analyse anisotropic samples. However, Jones matrix ellipsometry is not suitable for analysing depolarising samples. Mueller matrix ellipsometry along with the description provided by the Stokes-Mueller formalism, where real-valued Mueller matrix ( M ) elements connect the real-valued Stokes (S ) vector before and after sample interaction, can be used to characterise any sample (Equations (4.11) and (4.12)).

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Using the standard notation convention of [143], the Stokes vector with four real values is given by

II S0 ps  II S1 ps S , S II 2 45 45 S  (4.10) 3 IIRL

The quantities , , , and are the intensities of , , +45˚, -45˚, right and IIIIIp s 45 45 R I L p s left handed circular polarised light, respectively [143, 169].

Sout MS in , (4.11)

II  II  p s m11 m 12 m 13 m 14 p s     IIII p s  m21 m 22 m 23 m 24 p s       . IIm m m m II 45  45  31 32 33 34 45  45      m41 m 42 m 43 m 44 (4.12) IIRL out IIRL in

The relationship between elements of the Jones and Mueller matrices can be represented by the set of equations presented in Equation (4.13) , where  represents the complex conjugate of the reflection coefficients.

Similar to standard ellipsometry, an optical model is needed to describe the nominal structure of the film and to generate model Mueller matrix elements to compare with the measured elements. However, the MSE values of generalised ellipsometry (Equation (4.14)) on anisotropic samples cannot be directly compared with the MSE of best match models in standard ellipsometry.

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1     m11  rpp r pp  r ss r ss  r sp r sp  r ps r ps  2 1     m12  rpp r pp  r ss r ss  r sp r sp  r ps r ps  2

 m13 Re rpp r sp r ss r ps 

 m14 Im  rpp r sp r ss r ps 

1     m21  rpp r pp  r ss r ss  r sp r sp  r ps r ps  2 1     m22  rpp r pp  r ss r ss  r sp r sp  r ps r ps  2  m23  Re rpp rsp rr ss ps   m24 Im  rpp r sp r ss r ps   m31 Re rpp r ps r ss r sp   m32 Re rpp r ps r ss r sp   m33 Re rpp r ss r ps r sp   m34 Im  rpp r ss r ps r sp   m41  Im  rpp r ps  r ss r sp   m42  Im  rpp r ps  r ss r sp   m43  Im  rpp r ss  r ps r sp   (4.13) m44 Re rpp r ss r psrsp .

The data set for generalised ellipsometry is comparatively large compared to standard ellipsometry, as generalised ellipsometry analysis takes into account the closeness of the best match model to the measured spectra as a function of the sample rotation angle and the angle of incidence. As a result, regression analysis is used to correlate between the measured and model-generated Mueller matrix data that match as closely as possible.

2 S 44c 2 1 MMkl,, j kl j GE M    . (4.14)  Mkl 16SKj1 k  1 i  1  j

In Equation (4.14), the parameter S again represents the number of measured points and K is the number of model parameters, as defined for the MSE in standard ellipsometry.

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The optical polarisation response of a material can be represented by its optical constants as described earlier and is highly dependent on the electromagnetic response of that material. For a linear non-magnetic medium, the relationship between the electric field ( E ) and dielectric polarisability function ( D ) [109] can be written as

D  0εE, (4.15) where 0 is the free space permittivity. In an isotropic material, and are always parallel to each other as the dielectric function (ε ) becomes a single/scalar value as discussed earlier. However, for an anisotropic material, and are not necessarily parallel to each other [169]. Therefore, the optical response of an anisotropic material can be described by a three-dimensional dielectric tensor εε x,, y z . In order to address the

ε x,, y z correctly, let the plane of incidence [ xz, ] and the sample surface [ xy, ] set a right- handed Cartesian system ( x,, y z ) with the origin at the sample surface. The optical constants of a material in Cartesian laboratory coordinates ( ) can be expressed as a complex-valued second rank tensor , based on the sample Cartesian coordinates

( abc,, ) and real-valued Euler angles (,,   ) [143]. The corresponding schematic is depicted in Figure 4.15 and the associated coordinate transformation is provided in Equations (4.16) and (4.17).

Figure 4.15: Schematic showing the Euler angles ( ) and the orthogonal rotations as provided by rotational matrix A . Page | 72

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The coordinates ( abc,, ) and ( x,, y z ) refer to the sample Cartesian and laboratory coordinate systems, respectively. The First rotation is  around the z axis, then the coordinate system is rotated by  around the new x axis and finally rotated by  around the c axis [143].

   xx xy xz a 00      1, εx,, y z yx yy yz A 00b A (4.16) 00 zx  zy  zz c

cos cos cos sin sin sin  cos  cos sin cos sin  sin  A cos sin  cos cos sin sin  sin  cos cos cos sin  cos . sin sin  sin cos  cos

(4.17)

The optical constants εabc,, which are derived from the ellipsometry data modelling at a particular wavelength are diagonal in an orthogonal system. The Euler angle indicates how much the optical axis of the sample is tilted away from the axis. Moreover, the Euler angle shows the orientation of the a axis from axis, and the orthogonal rotation matrix A transforms the interior sample coordinates into lab coordinates where the ellipsometry measurements are taken. A detailed description of the rotation of coordinate systems using Euler angles can be found elsewhere [143].

4.7.2 Results

The Mueller matrix formalism was used to address the anisotropy of the Ag OAD86° 100 nm nanostructured film, with a polariser-compensator-sample-analyser ellipsometer which is capable of measuring 11 out of 16 Mueller matrix ( M ) elements normalised to m11 . The measurements were performed using a manual rotation stage at 37 in-plane clockwise rotation angles  from 0° to 360° with respect to the direction of the deposition vapour flux, and at three different incidence angles of 50°, 60° and 70°. The Ag OAD86° 100 nm samples deposited on microscope glass (glass OAD86°) and single-side sandblasted sapphire (sapphire OAD86°) were used for the analysis. The bare glass and sapphire substrates were modelled with a Cauchy dispersion model as discussed in Section 4.5.2.

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4.7.2.1 Glass OAD86°

Note: This section has been modified from the following paper.

M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Effective optical constants of anisotropic silver nanoparticle films with plasmonic properties”, Optics Letters, 2016. 41(23): p. 5495-5498.

The optical constants of a uniaxial material in Cartesian laboratory coordinates can be expressed as a complex-valued second rank tensor (ε  abc,,   ) in Cartesian sample coordinates and real-valued Euler angles (, ) when a =b [169]. Therefore, the corresponding complex dielectric function has two identical ordinary components  a =b and one extraordinary component c .

A homogeneous uniaxial model with fitted parameters of the Euler angles ( ) and film thickness was used to represent the nanostructured Ag-air layer. Moreover, the layer optical constants were parameterised with four generalised oscillators including three Gaussian oscillators and a Tanguy oscillator. The latter ensures KK consistency [180] and avoids some noise in the experimental data at the far ends of the spectrum. The physical parameters of the uniaxial model were optimised by nonlinear regression analysis in order to achieve the best match between the measured data and the model-generated results [143, 169]. The Equations (4.18) and (4.19) show the mathematical definitions of Gaussian and Tanguy oscillators that were used for the parameterisation. It has been experimentally shown by several groups that Gaussian oscillators provide a better fit for ellipsometry data of noble metal films than Lorentz oscillators to represent the LSPR [128, 169, 181].

As a function of photon energy, the Gaussian-oscillator-model-based dielectric function is expressed by

22 EE  EE  nn    EEEE       i   A  nn       i expnn  exp   , Gaussian 12n  nn     

(4.18) where n is the oscillator number, An is the peak amplitude of  2 , En is the centre energy,

Bn is the broadening or FWHM when  nn B 2 ln 2  and  is a convergence series that follows the KK consistent line shape for 1 . Page | 74

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The Tanguy oscillator is used to represent both bound and unbound states of electrons and is represented by

AR      nn       , Tanguy 12i g E iBn   g  E iBn  20 g    E iBn 

(4.19) where n is the oscillator number, An is the amplitude, Rn is the unscreened binding energy,

Bn is the broadening and g   is the screening factor. A detailed description of generalised oscillators can be found elsewhere [170, 182].

For the Ag OAD86° 100 nm sample deposited on glass, the good agreement between the model and measurements for the diagonal and non-zero off-diagonal elements of the Mueller matrix are shown in Figure 4.16.

Figure 4.16: Experimental and best match generalised ellipsometry data versus sample rotation angle for AOI of 50°, 60° and 70° at  = 514 nm for Ag OAD86° 100 nm film deposited on glass.

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As can be seen in Figure 4.16, four pseudoisotropic orientations were observed at   0°,

90°, 180° and 270°, thus validating the uniaxial approach [125, 183]. Pseudoisotropic orientations are the sample rotation angles at which off-diagonal elements of the Mueller matrix are zero. Depolarisation is known to occur when measuring inhomogeneous, nonuniform and very rough samples [107, 169, 184-186] due to the incoherent superposition of light rays with different polarisation states [122]. Since the Ag OAD86° 100 nm film has nanostructures that are much smaller than the probing wavelength, no significant sample depolarisation was observed. This can be confirmed by the values of m22 , which are close to one [187] as shown in Figure 4.16.

The three Gaussian oscillators and one Tanguy oscillator that were used to model the effective optical constants in the ordinary and extraordinary directions are shown in Figure 4.17. The dominant Gaussian oscillators 1 and 2 (centred at 2.2 eV and 2.5 eV, respectively) correspond to the LSPR, while the Tanguy oscillator accounts for the interband transitions above about 3.8 eV [59]. The third Gaussian oscillator centred at 3.4 eV is typically associated with the volume (or bulk) plasmon resonance [125]. The small deviations between the best fit dielectric constants and the Gaussian oscillators may be associated with higher-order effects [21] due to shape deviations from spherical symmetry [188].

Fitting data across a wide spectral range and three different incidence angles increased the accuracy of the model, reducing the MSE to a value of 5.7 and the best match model corresponds to an effective film thickness of 21.40 ± 0.04 nm. The effective optical constants derived from the uniaxial model are plotted as a function of wavelength in Figure 4.18 a) for fields polarised in the ordinary a (=b ) and extraordinary c directions of the sample coordinates.

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It is interesting to note the similarity of the appearance of results between Figure 4.13 and Figure 4.18 a), where Figure 4.13 corresponds to the pseudoisotropic orientation of the Ag OAD86° 100 nm with no mode transformations. However, the numerical values of refractive index and extinction coefficient are significantly smaller for the uniaxial model than for the pseudoisotropic approximation. The effective optical constants shown in Figure 4.18 a) are the average response of many nanoisland structures within the film in the probe area, including the interactions between particles. Further, the optical axis c of the nanostructures is inclined by  =83.41° ± 0.06° from the surface normal z towards the direction of the vapour flux. The Euler angle  =90.68° ± 0.01° describes the orientation of

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the a axis from the x axis and depends trivially on the accuracy of the initial sample position during the measurements.

It is clearly seen that the effective optical constants of the Ag OAD86° 100 nm film depart substantially from the planar Ag 100 nm film (Figure 4.9). The LSPR peaks of the Ag OAD86° film are observed at 500 nm and 470 nm in the extinction coefficients for both the ordinary and extraordinary axes, respectively. Although the films appear isotropic in the SEM images Figure 4.4 c) and Figure 4.11, their anisotropic uniaxial properties confirm that the nanoislands deviate from a hemispherical shape toward a truncated ellipsoid [189] as shown in Figure 4.18 b). Even at the initial stage of the nanoisland formation, it appears that the islands grow preferentially toward the incoming vapour flux, due to the geometric shadowing effect [76].

Interestingly, the observation of a plasmon resonance along each of the principal axes is in contrast to previous reports. For similar films deposited at normal incidence, Oates et al. found that the out-of-plane dielectric function of their Ag nanoparticles was similar to bulk Ag with a slightly increased broadening as shown in Figure 2.15 [125]. The Ag OAD86° 100 nm thin film described here does not exhibit bulk-like features along any principal direction. Ranjan reported double-localised plasmon resonance peaks due to the bimodal distribution of the nanoparticles [190]. Even in the absence of visual bimodal distribution in the Ag OAD86° 100 nm film, two significant LSPR-like oscillators are clearly seen in the uniaxial model for both the in-plane and out-of-plane dielectric functions (Figure 4.18 a). Hence, the observed anisotropy is likely to be traced back to small changes in particle shape/size and inter-particle spacing, which influences the coupling between the nearest neighbours.

4.7.2.2 Sapphire OAD86°

In order to study the effect of the underlying substrate refractive index on the effective optical constants of thin nanostructured films, a Ag OAD86° 100 nm film was deposited on a sapphire substrate. The sapphire substrate has a relatively high refractive index compared with the glass substrate as shown in Figure 4.8. However, the particle sizes of Ag nanostructures on sapphire (Figure 6.29) are fairly small compared to the structures seen on the glass substrate (Figure 4.4 c and Figure 6.19). Nevertheless, the area coverage of nanoparticles was found to be 46.0% and is comparable to the value of 48.6% for Ag OAD86° 100 nm on glass (Figure 4.4 c). For a better comparison, the nanoparticle sizes and the gap distances were calculated using the ImageJ software as shown in Figure 4.19. The results Page | 79

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confirm the generally smaller size of nanoislands on sapphire compared to those on glass (Figure 4.14). Interestingly, the gap distance follows the same trend as glass OAD, having the larger inter-particle spacing parallel to the deposition vapour flux.

For comparison, three Gaussian oscillators and one Tanguy oscillator were used to parameterise the uniaxial layer, applying the same ellipsometry modelling steps as described earlier in the results for the Ag OAD86° 100 nm film deposited on glass. Interestingly, the centre energies, amplitudes and the widths of the three Gaussian oscillators shown in Figure 4.20 are significantly different to those of the Ag OAD86° 100 nm film on glass substrate, as depicted in Figure 4.17. The three Gaussian oscillators and one Tanguy oscillator that were used to model the effective optical constants in ordinary and extraordinary direction are shown in Figure 4.20. The dominant Gaussian oscillator 1

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centred at 1.8 eV and Gaussian oscillator 2 centred at 2.0 eV represent the LSPR. The Tanguy oscillator accounts for the interband transitions above about 3.8 eV and the third Gaussian oscillator centred at 3.3 eV is typically associated with the bulk plasmon resonance. These two oscillators are presented together with the two LSPR Gaussian oscillators in the generalised layer, similar to those of the glass OAD86°.

Chapter 4

The model-generated and measured Mueller matrix elements at a wavelength of 514 nm for the Ag OAD86° 100 nm film deposited on sapphire are presented in Figure 4.21. The slight variations observed in the fitting were unavoidable due to some zeros appearing in the measured Mueller matrix elements, which were later identified to originate from an ellipsometer/ software issue (D. Schmidt, personal communication, December 11, 2016 and G. Pribil, personal communication, January 11, 2017).

Figure 4.21: Experimental and best match generalised ellipsometry data versus sample rotation angle for AOI of 50°, 60° and 70° at  = 514 nm for Ag OAD86° 100 nm film deposited on sapphire.

The effective optical constants derived from the uniaxial model are plotted as a function of wavelength in Figure 4.22 for fields polarised in the a (= b ) and c directions of the sample coordinates. The best match model gives an effective film thickness of 14.02 ± 0.01 nm, which is lower than the effective thickness of glass OAD86° (21.40 ± 0.04 nm).

It should be remembered that the effective medium represents an equivalent uniform material that best approximates the effective optical properties of the film, rather than providing a physical measure of the average height of the metallic nanoparticles. Variations in the physical thickness could arise due to differences in surface diffusion on the different

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substrate materials and slight changes in the sample orientation with respect to the evaporation source [73, 179], but it is not clear how these physical differences would influence the ellipsometry model results.

From the uniaxial model, the optical axis c of the nanostructures is inclined by  =83.9° ± 0.3° from the surface normal z towards the direction of the vapour flux. The Euler angle  =87.74° ± 0.07° describes the orientation of the a axis from the x axis and depends trivially on the accuracy of the initial sample position as discussed earlier.

Figure 4.22: The effective optical constants of the Ag OAD86° 100 nm film deposited on sapphire, in the sample coordinates ( abc,, ) as extracted from the uniaxial optical model.

The LSPR peaks of the Ag OAD86° 100 nm film deposited on sapphire are observed at 565 nm and 545 nm in the extinction coefficients for both the ordinary and extraordinary axes, respectively. Noticeably, these LSPR peaks are red-shifted from the corresponding peaks of the Ag OAD86° 100 nm film deposited on glass (glass OAD86°). The best match model corresponds to a MSE value of 9.9 and suggests that the model could be further improved with the addition of more oscillators such as combination of Gaussian and Drude oscillators to represent different optical phenomena [128].

It is interesting to see the red-shift in the LSPR peak of sapphire OAD86° compared to glass OAD86° as shown in Figure 4.23 from average absorption measurements of films. The average absorbance of the samples was normalised to [0, 1] using OriginPro 9.1.

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Figure 4.23: Average absorbance measurements of Ag OAD86° films on glass and sapphire normalised to [0,1], together with the corresponding refractive indices of the underlying substrates.

A study by Bakhti et al. showed a linear dependence between the positions of the plasmon resonance peak of Ag nanoparticles and the local refractive index of the surrounding medium. A gradual increase in local refractive index led to a red-shift in the plasmon resonance peak and a broadening of the plasmon band [191]. The LSPR peak of periodic arrays of Ag nanoparticles fabricated by nanosphere lithography has been reported to red- shift by approximately 1 nm for a change of 0.005 refractive index units (RIU) of the supporting substrate [192]. Based on this level of sensitivity, the difference between the refractive indices of the glass and sapphire substrates can be estimated as 0.28 RIU for the 55 nm wavelength shift of the Ag OAD86° 100 nm films. This compares favourably with n = 0.25 based on the refractive indices at the sodium doublet wavelength.

The presence of an interface can be considered to form of an image charge of the metal nanoparticles across the film interface. The cause for the net charge distribution on a supporting dielectric substrate has been discussed, as the image effect of an induced polarised charge distribution on the nanoparticles due to the external electromagnetic field [193]. The position of the screened plasma frequency at which the real part of the dielectric function of metal nanoparticles becomes zero, substantially affected by this image effect, as the symmetry of the surrounding medium of the metal nanoparticles is different due to the underlying substrate [59]. Using FDTD modelling, Wu and Nordlander showed the polarisation dependent red-shifts in the plasmon resonance due to the image screening introduced by the supporting dielectric substrate [194]. Therefore the red-shift in the LSPR Page | 84

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caused by the underlying substrate is well understood. Nonetheless, it would be an interesting experiment to examine the contribution of the supporting substrate dielectric constant to the red-shift in the plasmon resonance peaks observed in the uniaxial model, using a series of transparent substrates with different refractive indices. This however was beyond the scope of this thesis.

Note: Parts of this Section have been modified from the following paper.

M. N. M. N. Perera, W. E. K. Gibbs, S. Juodkazis, and P. R. Stoddart, “Wavelength and refractive index dependence of the geometrical enhancement in surface-enhanced Raman scattering”, Journal of Raman Spectroscopy, DOI: 10.1002/jrs.5190.

4.7.2.3 Near-side and far-side ellipsometry of Ag OAD86°

In this work, a preliminary comparison of the effective optical constants of Ag OAD86° 100 nm film was performed, taking into account the sample orientation (near-side and far- side, Figure 2.11) during ellipsometry measurements. A customised holder was used allowing ample room to rotate and flip the sample upside down during far-side measurements. A transparent UV-fused-silica substrate with a thickness of 6 mm (UQG optics, WFS 256) was used as the supporting substrate to fabricate a Ag OAD86° 100 nm film. A thicker supporting substrate was required in order to spatially separate the top and bottom surface reflections during far-side (reverse) ellipsometry measurements [195]. Microscope glass and sapphire substrates were too thin to allow near-side and far-side ellipsometry measurements of those substrates, due to the need to avoid interference from the unwanted reflections.

As described earlier, the Mueller matrix elements were recorded at 37 in-plane rotation angles for three incidence angles of 50°, 55° and 60° for the near-side and the far-side of the sample. For the far-side analysis, the z alignment of the ellipsometer was manually adjusted to only record the reflection from the fused-silica/film interface avoiding the top surface reflection from the air/fused-silica interface. It should be noted that the actual angles of incidence at the fused silica/film interface for far-side analysis are different to the above- mentioned angles of incidence due to light transmission through the air/fused-silica interface. This correction is automatically performed in the CompleteEASE software by applying Snell’s law at each interface. The CompleteEASE software first optimises the refractive index of the film during the fitting and then mathematically model the corresponding angle of incidence according to Snell’s law (G. Pribil, personal

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communication, April 14, 2017 and T. Tiwald, personal communication, July 15, 2017). The fused-silica substrate was modelled by a Cauchy layer and the nanostructured Ag film was modelled by a uniaxial layer. The order of the layers of the optical model were selected in CompleteEASE software according to the direction of the light source incident on the nanostructured Ag film during the measurement. The effective optical constants of the best match model for the near-side and the far-side of the sample are plotted against wavelength as shown in Figure 4.24.

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For the near-side of the sample, the best match model (MSE of 4.1) gives the Euler angles  = 91.1° ± 0.1°,  = 84.68° ± 0.02° and the effective film thickness =17.0 ± 0.3 nm. Running a similar uniaxial model for the far-side of the sample, the best match model (MSE 25.6) gives the Euler angles = 88.70° ± 0.01°, = 83.8° ± 0.1° and the effective film thickness =18.3 ± 0.1 nm. The slight changes in the Euler angles of the near-side data compared to those of glass OAD86° can be explained as arising due to the errors associated with positioning a thicker substrate under the evaporation source during oblique angle deposition.

These ellipsometric results also support the fact that the effective optical constants and hence the LSPR maxima of Ag OAD86° 100 nm films are highly sensitive to the supporting substrate of the film. The observed blue-shift in the LSPR maxima (Figure 4.24, near-side) for ordinary (475 nm) and extraordinary (465 nm) axes can be attributed to the lower refractive index of the fused silica substrate ( nD =1.46) when compared with the Ag OAD86°

100 nm films deposited on glass ( =1.52) and sapphire ( =1.77) at the sodium doublet wavelength.

There have been only a few reports of the far-side ellipsometric analysis of samples [173, 186, 195, 196] and to the best of our knowledge, nothing seems to cover far-side Mueller matrix analysis based on reflection measurements in the visible spectral range of a thin nanostructured metal film fabricated by OAD. For near-side and far-side ellipsometry measurements, it is reasonable to expect that the near-field interaction between the incident light and the surface plasmon charges and image charges inside the silica substrate will be different, as a result of differences in scattering cross sections, boundary effects arising from local driving fields and coupling efficiencies [100, 197-200].

Further, Okamoto and Yamaguchi showed that the resonance shifts and field enhancements originate from dipole-dipole interactions between a metal particle and its image in the substrate, and that the results are highly sensitive to the substrate refractive index [201]. Based on far-field reflectance and transmittance spectra of Ag nanoparticles on quartz, collected from both near-side and far-side illumination of the sample, Huang et al. showed that the far-side reflectance is always smaller than the near side due to asymmetric light reflectance from LSPR excited metal NPs, even though the transmittance spectra appeared to be exactly the same [95].

However, the significant differences observed in the effective optical constants for far-side and near-side measurements of the Ag OAD86° 100 nm film as shown in Figure 4.25, Page | 87

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together with the higher MSE for far-side suggest that a more detailed analysis is needed to fully understand these observations. Special care should be taken to confirm that the ellipsometry data are recorded on the same area of the sample to avoid any non- uniformities in thickness that may occur near the sample edges, as it could be an important factor in accounting for observed differences.

Figure 4.25: The difference between the uniaxial optical constants for the near-side and the far-side measurements of the Ag OAD86° 100 nm film, plotted against the wavelength.

A summary of the key results of Mueller matrix ellipsometric analysis performed in this Section is presented in Table 4.2.

Sample ID Silica OAD86° Glass OAD86° Sapphire OAD86° 1.46 1.52 1.77 nsubstrate (@ nD ) Effective thickness (nm) 17.0 ± 0.3 21.40 ± 0.04 14.02 ± 0.01 Euler  91.1° ± 0.1° 83.41° ± 0.06° 83.9° ± 0.3° angles  84.68° ± 0.02° 90.68° ± 0.01° 87.74° ± 0.07° Uniaxial 475 500 565 Ordinary ka LSPR peak 465 470 545 (nm) Extraordinary kc

Table 4.2: Summary of Mueller matrix analysis best match model results of Ag OAD86° 100 nm films deposited on substrates of fused-silica, glass and sapphire, respectively.

It is important to point out that the errors in the effective thickness and the Euler angles reflect the errors associated with the model fitting. The error due to the variability between samples and even the uncertainty for duplicated measurements on a single sample have not Page | 88

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been evaluated due to the time constraints. All of the samples used in this analysis were freshly deposited prior to the ellipsometry measurements, however a well-controlled experiment and a detailed analysis are highly recommended to study the dependence of the effective optical/dielectric constants of nanostructured Ag OAD films on shelf life and longer term exposure to air.

4.8 Conclusion

In this chapter, the effective optical constants of nanostructured Ag thin films fabricated on transparent substrates by the oblique angle deposition were studied using standard ellipsometry and Mueller matrix ellipsometry. While the standard ellipsometry helped to understand the general characteristics of the optical response of these plasmonic films, especially the Ag OAD80° and Ag OAD86° with a significant LSPR peak in the extinction coefficient, generalised ellipsometry within the extension of Mueller matrix analysis was used to carefully analyse the anisotropic optical response of the Ag OAD86° 100 nm film. The ellipsometric model-generated results confirmed that the Ag OAD86° 100 nm film exhibits a wavelength-dependent uniaxial optical response with an optical axis inclined towards the direction of the deposition vapour flux, even though the SEM images of the film show nanoisland like structures. Further, the Ag OAD86° 100 nm films deposited on sapphire, glass and fused-silica substrates confirmed that the effective uniaxial optical response of island-like layers is highly sensitive to the dielectric constant or the refractive index of the underlying transparent substrate. The LSPR peak of the extinction coefficient in the uniaxial model was red-shifted with increasing substrate refractive index.

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5 Theoretical Modelling of Geometrical SERS Enhancement

5.1 Introduction

In this chapter, the absence of optical constants of the metallic layer in the proposed “Fresnel model” for the geometrically-enhanced SERS signal by Jayawardhana et al. [20] is addressed using the ellipsometrically-determined effective optical constants. Prior to presenting the “extended Fresnel model”, a closer look at the recent models proposed in 2013 by both Funke and Wackerbarth [19] and Jayawardhana et al. [20] are presented as they appear to be the first attempts found in the literature to explain the additional enhancement in the SERS signal due to the excitation geometry of the sample. The optical response of nanostructured thin metal film samples is dramatically different to that of the bulk material and is most conveniently represented by the effective optical constants, which provide an average far-field description of the distribution of randomly oriented nanoparticles and their interactions with each other and surrounding media, and the incident light at the nanoscale. Therefore, the “Fresnel model” of Jayawardhana et al. [20] was extended by explicitly including the effect of the Ag OAD86° 100 nm nanostructured thin film, as represented by its complex refractive index. The effectiveness of the extended theory was then evaluated by comparison with the wavelength and angle of incidence dependent geometrical SERS enhancement (Chapter 6).

5.2 Fresnel equations

The Fresnel equations describe the polarisation-dependent amplitudes and phases of transmitted and reflected fields when light is partially reflected from and partially transmitted through an interface between two media with different refractive indices. Augustin Jean Fresnel first derived these equations in 1823 [202, 203].

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5.2.1 Parallel polarisation (p or TM polarisation)

Parallel ( p ) polarisation describes the situation when the electric field vector Ei of the incident light is parallel to the plane of incidence. The plane of incidence is that which contains both the surface normal and the direction of the incident light. The electric field vector components are shown in the Figure 5.1 where , Er and Et are the incident, reflected and transmitted electric fields respectively [202, 203].

Figure 5.1: Representation of polarisation. The sample plane is [ xy, ] and the plane of incidence is [ xz, ].

Relative to the incident field, the transmitted and reflected fields are given by [203]:

Etp 2nii cos t p  , (5.1) Eip n tcos i n i cos t

Erp nntcos i i cos t rp  , (5.2) Eip n tcos i n i cos t when i ,r and t are incidence, reflection and transmission angles respectively. t and r are known as the transmission and the reflection coefficients of the electric fields. The law of refraction (Snell’s law) can be applied at the interface to find these corresponding angles. For a better understanding of the definitions, additional notations have been used in Equations (5.1) and (5.2) to describe the incident ( i ), reflected ( ) and transmitted ( ) fields depending on their polarisation state. The permeability of two media is assumed to be equal to the permeability of free space, which is a valid approximation at optical Page | 92

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frequencies. ni and nt are the refractive indices of the first and second medium respectively. When i exceeds the critical angle c , the light transmitting to a second medium which is optically less than the first medium ( > ), is completely reflected at the interface. This optical phenomenon is known as the total internal reflection and the

1 nt corresponding critical angle is c  sin . ni

5.2.2 Perpendicular polarisation (s or TE polarisation)

Perpendicular ( s ) polarisation describes the situation where electric field vector of the incident light is perpendicular to the plane of incidence as shown in the Figure 5.2.

Figure 5.2: Representation of polarisation. The sample plane is [ xy, ] and the plane of incidence is [ xz, ].

Transmission (ts ) and reflection ( rs ) coefficients at perpendicular polarisation are given by the following equations [203]:

Ents 2i cos i ts  , (5.3) Eis n icos i n t cos t

Ers n icos i n t cos t rs  . (5.4) Eis n icos i n t cos t

At normal incidence, =0 and consequently t =0, therefore the Equations (5.1)-(5.4) can be simplified to the following: Page | 93

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2ni ts  , (5.5) nnit

nnit rs  , (5.6) nnit

ttps , (5.7)

rrps . (5.8)

For light normally incident on the interface from the first medium, the parallel and perpendicular polarisations are physically equivalent. However, in the definition of the positive direction of these fields in the usual derivation of the Fresnel relations there is an asymmetry. The phase change on reflection is typically included in the definition of the direction of the electric field in the reflected wave for the parallel polarisation and not included in the definition of the positive direction of the electric field for the perpendicular polarisation. It should be noted that the choice of the positive directions for the parallel polarisation is shown in Figure 5.1. In practice, it is the perpendicular polarisation that is usually assumed in normal incidence, as the sign of the field can be treated algebraically. The phase change of 180° on reflection when light travels from a less to more optically dense medium is shown with an explicit negative sign only by the definition of the equation of the perpendicular polarisation as explained below.

From air ( ni =1) to glass ( nt =1.5) interface; rs =0.2 and rp =0.2,

From glass ( =1.5) to air ( =1) interface; =0.2 and =0.2.

Since the intensity ( I ) is proportional to the square of the field amplitude, reflectance ( R ) and transmittance (T ) can be defined as follows, recalling that the transmitted intensity is now in a different refractive index medium to the incident intensity.

2 I E Rrreflected r  2 , (5.9) IEincident i

2 I ncos E n cos (5.10) Tttransmitted  t t t t t 2 . Iincident n icos i E i n i cos i

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5.2.3 Absorbing medium

For light travelling through and out of a finite film of material, the Fresnel equations can be used to represent the light reflection and transmission of that medium. The above Fresnel equations are applicable to an absorbing medium by replacing the (real) refractive indices by their complex counterparts [203]. A plane wave is assumed to be incident from a dielectric medium 1 onto the plane surface of absorbing medium 2 at an angle of incidence

1 as illustrated in Figure 5.3. The back boundary of the absorbing layer is another plane parallel surface with dielectric medium 3. The absorbing medium has a complex refractive index n2 represented by a real part and an imaginary part (Equation (2.19)).

Figure 5.3: Schematic of light incident onto a plane surface of an absorbing medium and the light reflection from and transmission through the next boundary.

The successive reflection and transmission amplitudes at the boundaries between medium 2 and the surrounding media 1 and 3 can be simplified using a converging geometric series and is found elsewhere [204]. Therefore, the simplified complex reflection coefficient at the top interface and transmission coefficient at the bottom interface of the absorbing medium are given by Equations (5.11) and (5.12) respectively [203, 205, 206].

r12 r 23 exp i  r  , (5.11) 1 r12 r 23 exp i 

t12 t 23 exp i 2  t  . (5.12) 1 r12 r 23 exp i 

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Each reflection and transmission coefficient in Equations (5.11) and (5.12) is dependent on the corresponding refractive indices of the media, angles of incidence, reflection and refraction, and the polarisation of the incident electric field, as can be seen in Equations (5.13)-(5.16). It should be noted that these equations give the amplitudes of the waves in each medium, not power coefficients.

The Fresnel coefficients for the perpendicular ( s ) polarisation are

nn1cos 1 2 cos 2 rs,12  , (5.13) nn1cos 1 2 cos 2

2n11 cos ts,12  , (5.14) nn1cos 1 2 cos 2 and for parallel ( p ) polarisation

nn2cos 1 1 cos 2 rp,12  , (5.15) nn2cos 1 1 cos 2

2n11 cos t p,12  . (5.16) nn2cos 1 1 cos 2

Similar expressions can be written for the reflection and transmission coefficients, rs,23 , ts,23 , rp,23 and t p,23 at the boundary between media 2 and 3. The permeability of all three media is assumed to be equal to the permeability of free space, which is a valid approximation at optical frequencies. The relationship between the complex phase factor

 , the complex refractive index and the complex refracted angle is given by

d  4 n cos  . (5.17)  22

The law of refraction (Snell’s law) can be applied at each interface to find the corresponding angles [203].

n1sin 1 n 2 sin  2 n 3 sin  3 , (5.18)

n2  n ik. (5.19)

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The angle 2 is complex and has a formal meaning only [207]. Several approaches of calculating a complex refraction angle based on the planes of constant amplitude and phase are discussed elsewhere [207-209].

5.3 Revisiting the current models

Two attempts at unfolding the theoretical basis of the far-side geometrical enhancement in SERS were reported in 2013 and have two different approaches as described below. For convenience, the near-side and far-side excitation geometries of the sample as depicted in Figure 2.11, is used to present both models in this summary.

5.3.1 The EM model by Funke and Wackerbarth

A theoretical model based on the electromagnetic enhancement mechanism was used to show the enhancement in SERS signal based on the illumination geometry for a spherical metal nanoparticle placed on a supporting substrate [19]. The dielectric function of the metal was represented by metal  12i  and the surrounding medium by  d . For the near-side excitation, the spherical nanoparticle was assumed to be surrounded by the medium air ( =1.00) and for the far-side excitation, it was considered to be a quartz

( =2.37) substrate. Equation (5.20) represents the proposed EM model of Funke and

Wackerbarth, where L and S denote the excitation and Stokes-shifted frequencies, respectively.

2222                          11L d  22L  S d  S   . (5.20) EM 2222    22                     11L d  22L   S d  S 

The model was applied to a Ag nanoparticle with a diameter of 45 nm and a Au nanoparticle with 60 nm. Figure 5.4 shows the enhancement (  ) predicted by the model for the EM nanoparticle in air and quartz for the nitrothiophenol vibrational mode at 1331 cm-1. The two maxima correspond to the laser and scattering wavelengths at which the denominator of Equation (5.20) is a minimum. The theoretical model showed a red-shifted enhancement for excitation in the quartz substrate, with an enhancement factor of 1.7 for Ag and 5 for Au

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compared to the case of air. The experimental results at 785 nm excitation were reported to be 1.4 and 2.5 for Ag and Au respectively.

This model suggests that the far-side enhancement is associated with the effect of the dielectric substrate on the interaction between the light and the nanoparticle. However, this simplified model of the EM enhancement treats the nanoparticle as it is immersed in the dielectric medium, where as in reality the dielectric environment is unchanged between near-side and far-side excitation. The simplified enhancement model also makes it conceptually difficult to extrapolate the results for different excitation and scattering frequencies, or to analyse different plasmonic film structures and angle of incidence [18]. Page | 98

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5.3.2 The Fresnel model by Jayawardhana et al.

A simple analytical model based on the Fresnel equations was proposed to represent the electric field amplitudes for near-side and far-side excitation of the sample at normal incidence [20]. Since the intensity is proportional to the square of the amplitude of the field, the relative enhancement was calculated as the square of the ratio of far-side SERS intensity

4 to near-side SERS intensity, based on the E approximation discussed in Section 2.3.

Equation (5.21) shows the proposed Fresnel model, where r12 and t12 are the reflection and transmission coefficients at the air-glass interface and t23 is the transmission coefficient for a wave travelling through the glass substrate towards air.

4 tt g. 12 23 (5.21) 1 r12

The model gives an enhancement factor of 2.01, while the experimental results showed approximately 3 times enhanced SERS signal at 514 nm for the far-side. Moreover, 3D-FDTD simulations were performed with FDTD Solutions software (Lumerical Solutions, Inc) representing two hemispherical Ag nanoparticles of 30 nm diameter and 15 nm height placed 6 nm apart on a SiO2 slab. The calculated electric field distributions associated with near-side and far-side excitation can be seen in Figure 5.5. The FDTD simulation results gave a relative enhancement factor of 2.16, assuming that the SERS signal was primarily arising from the hot spots.

Chapter 5

Similar to the Fresnel picture, the FDTD modelling suggested that an increased electric near- field at the dielectric boundary could be driving the increased far-side SERS signal. However, the Fresnel model ignored the presence of the absorbing metal film and considered propagation from air to substrate and vice versa only. On the other hand, FDTD is not good at evaluating an ensemble of random nanoparticles or even larger ordered array in practical sensing applications.

5.4 Extended Fresnel model

In both of the previously discussed theoretical models, the absence of a realistic representation of the nanostructured metal film appears as a limitation to our understanding of the far-side geometrical enhancement. In the scope of this thesis, the aim was set to explore whether treating the nanostructured metal film as an effective medium can provide a more accurate quantitative prediction of the observed geometrically- enhanced SERS effects. The Ag OAD86° 100 nm film deposited on a transparent dielectric substrate was used as the model system to represent the geometrical enhancement in the SERS signal. The wavelength-dependent effective optical constants of the Ag nanostructures and the thickness as determined by Mueller matrix ellipsometry (Section 4.7.2) were used to represent the nanostructured metal film in this extended Fresnel model. The wavelength- dependent refractive indices of the glass and sapphire supporting substrates were also used to represent the physical sample as accurately as possible. Figure 5.6 shows the near-side and far-side excitation geometries of the SERS substrate with the corresponding notations that are later used in the extended Fresnel model.

Figure 5.6: Schematic of a) near-side and b) far-side interrogation of the SERS substrate with the corresponding notations that are used in the extended Fresnel model. Page | 100

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For near-side illumination of the sample at normal incidence, light first travels through air (a), film (f) and then into the transparent substrate (s). Considering the continuity of the tangential components of the electric fields across the boundaries [203], the following equations were used to calculate the corresponding electric field amplitudes. The magnitude of the incident E-field was considered as 1 and the notations of i , r and t are used to represent the incident, reflected and transmitted electric field components, respectively, with:

EEEi r t , (5.22) at each successive interface. Then following [205] and Equation (5.12), the magnitude of the electric field at the film-substrate interface is Et near

 , Etinear t E (5.23)

taf t fs exp i 2  t  . 1 raf r fs exp i  (5.24)

The corresponding transmission and reflection coefficients at each interface can be   2na 2n f nnaf nnfs d calculated as taf  , t fs  , raf  , rfs  and  4 n f nnaf nnfs nnaf nnfs  by setting the incidence angle to zero in Equations (5.13) and (5.14). FDTD modelling suggests that the hot spots are generated at the film-substrate interface and are hottest in the narrow gaps between closely spaced nanoislands [4, 70, 210, 211]. Therefore the field intensities at the film-substrate interface were used to compare our calculations for near- side and far-side excitation. The total SERS field in the near-side case can therefore be approximated as the product of the locally enhanced incident and scattered fields [212, 213].

EEE     . (5.25) SERS near t exnear t sca near

The Stokes shift of an analyte at a particular excitation wavelength is relatively small compared to the excitation wavelength [214]. Hence, the corresponding effective optical constants of the metal layer at the incident and scattered wavelengths were assumed to be equal for the field calculations for a particular excitation wavelength as expressed by

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2 EE   . (5.26) SERS near t ex near

For far-side illumination of the film, light first passes through the air (a), then the transparent substrate (s) and finally into the film (f). Considering the continuity of the tangential electric fields across the boundaries as represented by Equation (5.22), the corresponding magnitude of the electric field at the substrate-film interface becomes

. Etifar E 1 r (5.27)

The reflection losses that occur at each interface are incorporated in

rsf r fa exp i  r  , (5.28) 1 rsf r fa exp i 

where rrsf fs and rrfa af . As can be seen in Equation (5.27), the incident field Ei on the substrate-film interface is now modified due to the phase shift that occurs at the air- substrate boundary

Ei  t as E i , (5.29)

2na where tas  . Similar to Equation (5.26), the total SERS field for the far-side nnas excitation geometry can be calculated as

2 EE   . (5.30) SERS far t ex far

The electric fields associated with the absorbing metal film at each interface were calculated in MATLAB R2012b (8.0.0.783) using the complex numbers that represent the real refractive index and the imaginary extinction coefficient of the film. Detailed calculations of the Fresnel reflection and transmission coefficients for a uniaxial material at normal incidence are discussed elsewhere [215, 216]. Figure 5.7 shows the corresponding magnitudes of the SERS fields (Equations (5.26) and (5.30)) when the incident electric field was polarised parallel and perpendicular to the ordinary axis of the uniaxial metallic film discussed in Section 4.7.2.1.

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As can be seen in Figure 5.7, for both polarisation directions, the magnitude of the SERS E- field is enhanced for far-side illumination of the film compared to near-side illumination and is highly influenced by the absorption in and reflection from the thin metal film when light propagates in and out through the film. It should be noted that the enhanced fields near 320 nm is characteristic of silver and occurs due to the lowest interband electron transitions. Therefore, based on Equations (5.26) and (5.30), the corresponding SERS field intensities can be calculated at the substrate-film boundary, so that the geometrical enhancement factor (g):

2 4 IEEt g.SERS far  SERS far  far (5.31) IE  E SERS near SERS near t near

The factor g represents the geometrical enhancement occurring due to the sample orientation and allows the dependence on excitation wavelength to be predicted.

Further, if the SERS field is assumed to propagate towards air, considering the signal collection optics in air (near the objective) for near-side and far-side excitation separately, the output field in air can be calculated using the Equations (5.32) and (5.33).

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ESERS near/ air E SERS near expi 2  t fa , (5.32)

(5.33) ESERS far/ air E SERS far t sa .

Therefore, the geometrical enhancement factor for the scattering collected in air, considering the corresponding intensity ratios is:

2 IESERS far// air SERS far air g.air  (5.34) IESERS near// air SERS near air

For the sake of simplicity, an average enhancement factor was obtained by averaging the corresponding enhancement factors for the excitation polarisation corresponding to the ordinary and extraordinary axes of the glass OAD86°. Figure 5.8 shows the extended Fresnel model predictions for the average geometrical enhancement factor (GEF) plotted against wavelength at the film-substrate boundary and at the air-side of the substrate facing the signal collection lens. The effective optical constants of Ag OAD86° 100 nm deposited on glass were used for the modelling as derived in Chapter 4.

Figure 5.8: The average geometrical enhancement plotted against excitation wavelength at the film-substrate boundary and at the air-side of the substrate facing the collection lens.

The extended Fresnel model predicted GEF consistently shows an increased far-side SERS signal compared to near-side with GEF >1 for the entire wavelength range of 300-1000 nm. The enhancement peak around 330 nm for the GEF curve for air side collection may be Page | 104

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explained by the characteristic absorption when light passes through the absorbing film and is only present for the near-side propagation path towards the collection lens compared to the far-side case. For both GEF curves, the highest enhancement factors are seen to be blue- shifted from the maximum of the absorbance spectra of the nanostructures (Figure 4.12). This blue-shift is believed to arise from interactions between the various components in the extended Fresnel model, including phase shifts at the boundaries and interferences within the thin metal island film and between components reflected at the various boundaries [95, 97, 217].

As explained earlier based on FDTD modelling results, it seems reasonable to assume that the SERS field is “hottest” at the film-substrate interface. Therefore, the best experimentally represented GEF can be considered as the curve predicted at the film-substrate interface. The experimental enhancement factors of 1.4 and 3 reported by Funke and Wackerbarth [19] and Jayawardhana et al. [20] were also found to follow the values on this curve at excitation wavelengths of 785 nm and 514 nm, respectively. For comparison, Figure 5.9 shows the average GEF calculated at the substrate-film interface with Equation (5.31) for Ag OAD86° 100 nm film deposited on glass (Glass OAD86°) and sapphire (Sapphire OAD86°). The effective optical constants of the glass OAD86° and sapphire OAD86° samples were discussed in Section 4.7.2.

Figure 5.9: The wavelength-dependent average geometrical enhancement factor for far-side versus near-side excitation, calculated at the film-substrate interface by the extended Fresnel model.

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For both samples, the maximum peaks of the theoretical enhancement factor curves in Figure 5.9 are blue-shifted from the maxima of the absorbance spectra of the nanostructures (Figure 4.23). This blue-shift arises due to interactions between the various components in the extended Fresnel model as explained previously. Additionally, the theoretical model for sapphire OAD86° predicts a broader enhancement factor curve than the glass OAD86° within the visible wavelength region of the EM spectrum. However, it is interesting to note that the inclusion of the metal film in the extended model leads to a prediction that the geometrical enhancement factor of the sapphire substrate is comparable in magnitude to that of the glass substrate, whereas the simplified model of Jayawardhana et al. [20] predicted that sapphire would outperform glass by approximately 35%. Interestingly, the peak of the wavelength-dependent geometrical enhancement factor curve for sapphire is red-shifted compared to that of the glass substrate.

In order to model the angle of incidence dependent GEF, the Equations (5.24) and (5.28) were modified by including the angle-dependent reflection and transmission coefficients as previously expressed by Equations (5.13) and (5.14). Figure 5.10 shows the angle- dependent magnitude of SERS fields for the near-side and far-side excitation, and the corresponding geometrical enhancement factor at the film-substrate interface, as predicted by the extended Fresnel model at excitation wavelengths of 514 nm and 633 nm for Ag OAD86° 100 nm film on glass.

Figure 5.10: Angle dependence of the SERS field for near-side and far-side and the corresponding geometrical enhancement factor.

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The incidence polarisation was considered to be parallel to the ordinary axis of the uniaxial model of Ag OAD86° for simplicity and the SERS signal to be modelled accurately for the near-side and far-side excitation. The angle of incidence was defined as the angle between the substrate normal and the incident laser light as shown later in Figure 6.33. As can be seen in Figure 5.10, the magnitude of the SERS field decreases for both the far-side and near- side excitation as the angle of incidence increases and follows a cosine-squared curve shape. A similar shaped curve has been previously reported in [218] for the angle-dependent SERS field under near-side excitation. Interestingly, the extended model predicted geometrical enhancement factor tends to increase rapidly for higher incidence angles and depends on the excitation wavelength. However, the utility of this effect will be limited significantly by the optical setup and reduced signal intensity when it comes to practical applications.

Note: Parts of this Section have been modified from the following paper.

5.5 Conclusion

In this chapter, the geometrically-enhanced SERS signal is theoretically modelled by applying an extension to the “Fresnel model” proposed by Jayawardhana et al. [20]. The absence of the optical constants in the Fresnel model was addressed by including the effective optical constants of Ag nanostructured films as determined by the Mueller matrix ellipsometry. The wavelength and angle of incidence-dependent Fresnel reflection and transmission coefficients represented by complex numbers were used for the corresponding electric field calculations for the near-side and far-side excitation geometry of the SERS substrate. The extended Fresnel model allows the wavelength and angle of incidence dependent geometrical enhancement factors in SERS to be calculated and to the best of our knowledge is the first attempt to use the effective optical properties of nanostructured films to do so. The extended Fresnel model predicted GEF consistently shows a higher enhancement factor for the far-side excitation over the wavelength range of 300-1000 nm and can be used for different SERS sensing applications in the UV, visible, IR and far IR of the EM spectrum. On the other hand, the angle dependence predictions suggest that there is likely to be limited benefit in using oblique angle of incidence to maximise the

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performance of the far-side excitation geometry. In the following Chapter, experimental results are presented to test these new theoretical predictions.

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6 Experimental Study of Geometrical SERS Enhancement

6.1 Introduction

Surface-enhanced Raman scattering is an important analytical technique that allows for label-free detection of molecules even at a single molecule sensitivity. Several experimental reports of the geometrically-enhanced SERS signal were discussed in Section 2.4. However in most of those studies, the results were limited to a single excitation wavelength for a particular SERS substrate. The optimisation of the geometrically-enhanced SERS signal can be achieved only if the key contributing factors are well understood. Therefore, this Chapter describes the experimental efforts undertaken in order to improve the fundamental understanding of the effect.

The experimental work begins with Raman experiments for system calibration followed by SERS experiments to study the geometrically-enhanced signal. The key factors studied in the Raman experiments included the dependence of the scattering on solid angle, excitation laser power, Raman microscope depth-of-field and source polarisation (Section 6.2). In the SERS experimental section, the repeatability of the Ag OAD86° 100 nm substrate together with the SERS signal dependence on the solid angle, depth-of-field and excitation laser power were further studied (Section 6.3). The heart of this experimental chapter is the geometrical SERS experiments section that is comprised of a comparative study between the near-side and far-side SERS signals that provide the experimental GEF under a range of conditions. Thus, an extensive study of the geometrically-enhanced SERS signal is presented in terms of the key factors of excitation wavelength, substrate dielectric material, angle of incidence, polarisation and metal layer thickness (Section 6.4). In addition, a Raman mapping of the near-side and far-side of the Ag OAD86° 100 nm SERS substrate, and a chemical composition analysis performed based on X-ray photoelectron spectroscopy of the transparent dielectric substrates and Ag OAD86° films are presented in this Chapter.

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6.2 Raman experiments

Prior to performing any SERS analysis on Ag OAD films, it was necessary to understand some fundamentals of Raman scattered light. A standard Si sample which is known to be an approximately angle-independent Raman scatterer was used for the initial measurements. The baseline-to-peak height of the Si Raman peak centred at 520 cm-1 was obtained from spectra recorded in the static scanning mode of the Renishaw inVia Raman spectroscope. In this section, the dependence of the Raman signal on solid angle, excitation laser power, polarisation direction and depth-of-field are presented.

6.2.1 Raman signal dependence on solid angle

Raman scattering is a relatively weak optical effect as discussed earlier and generally occurs evenly in all directions. The fraction of light that can be collected is normally determined by the solid angle. The solid angle (  ) and the numerical aperture (NA) of an objective holds the relationship of

2 NA  2 1  cos    2   1  1  , (6.1) n  where  is the half angle of the maximum cone of light that is captured by the lens, NA  nsin and n is the refractive index of the medium, normally air. The maximum possible angle that can be is 90° that in turns gives the collection of light a complete hemisphere of 2π steradians.

After exciting the standard Si sample through each objective, the Si peak counts at 520 cm-1 were used for the analysis. The excitation wavelength was selected as 514 nm and ten Si spectra were recorded across the sample for each objective. The peak heights were averaged during the analysis. The list of available objectives in the Renishaw inVia Raman system is given in Table 3.3. The spectra were corrected for laser power and the acquisition time. The laser power measured after each objective was considered as the excitation power at the sample and was significantly lower for the higher NA objectives.

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Figure 6.1: Raman scattering signal dependence on the solid angle. The error bars mark the standard deviation (SD) of ten measurements at different locations of the Si wafer.

As shown in Figure 6.1, a linear dependence of the scattered Raman signal versus the solid angle was observed. When Raman scattering is approximately isotropic for the backscattering geometry, the maximum solid angle is equal to 4π steradians. Considering the collection geometry in the Renishaw inVia Raman system, the results can be explained as a ratio of objective solid angle versus 4π. Therefore, the 5 and 100 microscope objectives collect only 0.36% and 23.6% of the total Raman scattered light, respectively. Due to the limitations of the coupling optics, the above explained numbers show that most of the Raman scattered light does not reach the detector.

6.2.2 Raman signal dependence on excitation laser power

Equation (2.7) shows that the total intensity of the Raman signal depends linearly on the excitation power of the laser. The Si peak counts at 520 cm-1 were recorded at five different locations on the sample using the 5 lens for three seconds exposure and three accumulations. The power of the 514 nm laser line incident on the sample was controlled from 1% to 100% during the measurements using the WiRE software to investigate the Raman signal dependence on the excitation power of laser. The total power (100%) measured after 5 lens using the laser power meter was recorded to be 11.1 mW.

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Figure 6.2: Dependence of Si peak counts at 520 cm-1 on the excitation laser power. The dash line marks the line of best fit with slope ~1.

The results depicted in Figure 6.2 show that an increase of the excitation laser power leads to a linear increase in the Si peak counts and agrees with the Equation (2.7), when the number of molecules and the Raman cross section remain the same during the described experimental conditions. The experiment was repeated with other objectives in the Renishaw system and the Si peak counts are shown in Figure 6.3, normalised against the laser power and acquisition time.

Figure 6.3: Comparison of Si peak intensity (corrected for the excitation laser power and acquisition time) with the excitation laser power for all objectives available on Renishaw system. Page | 112

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As might be expected, the power corrected Si peak intensities at different percentages of incident laser power holds an approximately constant relationship for each objective. The slight variabilities in the measurements account for the standard deviation of the measurements. Considering the results of both Figure 6.2 and Figure 6.3, it can be concluded that the Raman signal from an approximately angle-independent scatterer depends linearly on the incident laser power and collection aperture for any objective lens.

6.2.3 Raman depth-of-field

In order to test the confocality of the Raman microscope, silicon spectra were acquired using an excitation wavelength of 514 nm for different objectives. The z-scanning control of the motorised stage of the Renishaw system was varied from the sample focal plane in the upward and downward directions in 2 μm steps. Prior to the start of each series of measurements under each objective, the laser light was finely focused onto the Si sample using the video camera with the laser spot enabled and this position was set to be z=0. Figure 6.4 shows the Si peak intensity at 520 cm-1 as a function of depth.

Figure 6.4: Comparison of depth profiles of objectives including 5, 20, 50L, 50 and 100.

The solid line represents a Gaussian fit [219] to the set of data points associated with each objective. Everall used Lorentzian fits to represent the depth profiles of high NA objectives as the wings of the fit decay relatively slowly compared to those of the Gaussian [220].

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However, the Gaussian fit is satisfactory for current purposes, where the centre and width of the profile are of particular interest.

As can be seen in Figure 6.4, the full width at half maximum (FWHM) indicates the depth of focus of the confocal sampling arrangements [221]. The maximum Si peak intensity is seen when the sample surface is located in the focal plane of the objective (z=0) for the higher magnification lenses of 50L, 50 and 100. The Gaussian fit indicates that the peak intensity significantly drops off with displacement of the Si sample surface from the focal plane, exhibiting a good confocality for high magnification objectives. The centre of Gaussian deviation from z=0 is thus an indication of focusing accuracy. On the other hand, the Raman depth profile is highly sample dependent due to the fact that use of thicker substrates will generate out-of-focus rays compared to thin substrates [220].

The depth-of-field (DOF) of an objective indicates the axial resolving power that is generally measured parallel to the optical axis and can be calculated [222]:

 DOF . (6.2) 2 NA2

Figure 6.5: Schematic of depth-of-field ranges of high and low NA objectives. Reproduced from https://www.microscopyu.com/microscopy-basics.

Therefore, the DOF for high confocal of higher magnification lenses of 50L, 50 and 100 was found to be less than 1 m, whereas for the lower magnification lenses of 5 and 20 the values were 17.8 m and 1.6 m, respectively (Table 3.3). It should be noted that the Renishaw system is optimised for the higher NA lenses for Streamline Raman mapping and the manufacturer highly recommends the use of higher NA lenses for any analysis Page | 114

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(O. Milikofu, personal communication, December 1, 2014). Therefore, care was always taken to finely focus the laser beam onto the sample for all experiments to avoid the loss of Raman scattered signal and reduce measurement variability due to incorrect focusing of the laser beam.

6.2.4 Raman signal dependence on the source polarisation

The Renishaw inVia Raman system has three different polarisation settings named as normal, orthogonal and circular polarisation. The source polarisation was defined when parallel to x as “normal” and parallel to y as “orthogonal”, respectively (Figure 4.18 b). The source polarisation can be controlled by the WiRE software with automated insertion of half-wave and quarter-wave plates in the incident beam path of the spectroscope for orthogonal and circular polarisation states, respectively. Unless specified, the source polarisation was selected to be normal for all experiments in this thesis. Figure 6.6 shows the wavelength-dependent Si peak intensities recorded for normal and orthogonal polarisations. Peak intensities were normalised by power measured at the sample in each case. The results suggest that the scattered Raman signal is polarised and the scattering intensity is also wavelength-dependent. However, the polarisation direction of the scattered Raman signal is not necessarily the same as the fixed polarisation direction of the external incident electric field and highly depends on the orientation of the scatterer, such as the symmetry of molecular and crystal structures in space [28].

Figure 6.6: Si peak intensity for normal and orthogonal polarisation states of the laser source plotted against the excitation wavelength. Page | 115

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The polarisation state of the scattered Raman signal was investigated by setting a polarisation filter before the entrance slit (Figure 3.7) in the Renishaw Raman spectroscope. Firstly, the Si peak counts at 520 cm-1 were recorded for the normal and orthogonal polarisation states of 514 nm excitation as controlled by the WiRE software without a polarisation filter attached before the entrance slit. As mentioned earlier, source polarisation was defined when parallel to x as “normal” and parallel to y as “orthogonal”, respectively (Figure 4.18 b). A half-wave plate has been automatically set in the beam bath when the polarisation state changed from normal to orthogonal. Secondly, the Si peak counts were recorded for both source polarisations when the optical axis of the polarisation filter was set horizontally and vertically in the scattered beam path.

The results are shown in Table 6.1 and show the scattered Raman signal preferentially holds the same polarisation as the source polarisation. The polarisation efficiency (ratio of orthogonal + horizontal versus normal + vertical polarisation) of the scattered Raman signal on the grating for 514 nm can be estimated as 0.44, assuming the difference is all due to the grating [223], whereas crystal orientation may also matter with different polarisabilities of bonds in multicystalline silicon, given the slight changes observed in Si peak shifts [224]. The extended experimental results for 488 nm and 633 nm excitations showed that the grating polarisation efficiency is wavelength-dependent with values of 0.95 and 0.31 respectively.

Status of the polarisation Laser polarisation normal Laser polarisation orthogonal filter (PF) in the Peak Intensity Width Peak Intensity Width scattered beam path centre (Counts centre (Counts (cm-1) s-1 mW-1) (cm-1) s-1 mW-1) No PF 520.9 141.9 3.26 520.4 61.3 3.32 PF optical axis horizontal 520.8 1.1 3.78 520.8 46.4 3.25 PF optical axis vertical 520.4 105.5 3.38 520.3 1.6 3.66

Table 6.1: Polarisation-dependent Raman peak intensity for Si at 520 cm-1.

The above-discussed preliminary Raman experiments based on the Si peak intensity were gathered to characterise “baseline” performance of the system and to help minimise any ambiguities in the SERS experiments with Ag OAD films. In some cases this allows more firm conclusions to be drawn, especially in the experimental section of the geometrically- enhanced SERS signal.

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6.3 SERS experiments

6.3.1 Repeatability of OAD fabrication

According to the previous work of [225] it has been found that higher sample-to-sample reproducibility was achieved when the OAD substrates were deposited at a rate of 0.03 ± 0.01 nm s-1. Therefore, to check how successfully the deposition rate could be controlled by manual adjustment of the applied current to maintain a deposition rate at 0.03 nm s-1, ten different coating cycles of Ag OAD86° 100 nm films were compared. The deposition time in seconds and the film thickness in nanometres were noted down as indicated on the digital display on the Emitech turbo evaporator. A line of best fit for the graph of film thickness versus deposition time was used to calculate the deposition rate. Figure 6.7 shows the linear fitting results for ten coating cycles indicating the deposition rate as determined by the slope of the graph of film thickness versus deposition time and the correlation coefficient R2.

Figure 6.7: Reproducibility of Ag OAD86° 100 nm films.

Even though a considerable effort was taken to precisely control the deposition rate manually, Figure 6.7 shows the variability of the deposition rate across different coating cycles. No apparent correlation between the deposition rate and goodness of fit (R2) was observed. The reproducibility of the deposition rate was found with an accuracy of 0.03 ± 0.01 nm s-1. Page | 117

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6.3.2 SERS analytes

Many molecules show little or no attraction to metallic surfaces and do not generally exhibit any measurable SERS response. It is essential that molecules are either chemisorbed or physisorbed to the surface of the metallic nanostructures in order to being enhanced by the confined electromagnetic fields near the metallic surface. Frequently described target molecules in the literature are thiols, amines and aromatic macrocycles [226].

6.3.2.1 Thiophenol

Thiophenol (also known as benzenethiol, phenyl mercaptan or mercaptobenzene) is a foul- smelling colourless liquid that has the chemical formula of C6H5SH (Figure 6.8). As reported, thiophenol is a commonly studied SERS active molecule due to the strong covalent bonds between the sulphur and noble metals through loss of the sulfhydryl hydrogen (-SH) allowing it to form stable monolayers [226]. A study by Li et al. showed the adsorption behaviour of thiophenol on gold nanoparticles by obtaining the FT-Raman spectra of pure thiophenol and thiophenol adsorbed on Au NPs [227]. In addition, Tripathi et al. experimentally showed the pH dependence of the thiophenol binding into gold via chemisorption and physisorption processes [226]. Moreover, it has been shown experimentally and theoretically that there are slight changes in the vibrational mode frequencies between Raman spectra of neat thiophenol and SERS spectra of metal-bound thiophenol as summarised in Table 6.2 [228-230].

For most of SERS measurements discussed in this thesis, thiophenol was used as the analyte. OAD substrates were submerged in 10 mM thiophenol (Sigma-Aldrich,>99%) ethanolic (Merck Millipore, >99.9%) solution for 20 minutes. Thiophenol was always handled with care as it is very toxic, flammable and reactive. Page | 118

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Neat thiophenol SERS thiophenol Vibrational assignment (cm-1) (cm-1) 412 418 C–S stretching 697 693 C-H out of plane deformation 892 890 C-H out of plane bending 915 Not observed S-H in plane bending 999 997 S-H bending + in plane ring deformation 1024 1021 In plane ring deformation 1091 1072 C-S stretching + in plane ring deformation 1580 1572 C–C stretching

Table 6.2: Comparison between normal Raman peaks and SERS peaks of thiophenol.

The thiophenol solution was prepared by diluting 103 μl of thiophenol in 100 ml of spectroscopic grade ethanol. In this process, a self-assembled monolayer (SAM) of thiophenol is formed around the silver particles via S-Ag bonding. A self-assembled monolayer indicates that each sample tested will contain the same area density of molecules, provided that the nanoparticle size, structure and shape is consistent. After the reaction period, the samples were removed and rinsed in ethanol to remove unbound thiophenol and solvent, and finally dried in nitrogen. Figure 6.9 shows the normalised counts of the Raman spectra of pure thiophenol (Sigma-Aldrich, >99%) measured inside a glass vial and SERS spectra of 10 mM thiophenol ethanolic solution on Ag OAD. The central positions of the main peaks of the two spectra were determined using the peak analyser function on Origin Pro 9.1. The resulting peaks show good agreement with the peak position in Table 6.2, with an average deviation of  1 cm-1.

Figure 6.9: Measured peak positions for Raman and SERS spectra of pure thiophenol and thiophenol SAM excited with 514 nm. Page | 119

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For a comparative study, SERS spectra were collected using all excitation wavelengths in the Renishaw Raman system. The peak positions and FWHM of the four main peaks were determined by fitting a Gaussian peak function and the results are summarised in Table 6.3. Aggarwal et al. determined the SERS line widths of thiophenol SAM on silver coated SERS substrates and discussed the difference between the Raman line widths of neat thiophenol [230]. The two contributing factors were identified as the fact that the SAM of thiophenol has molecules that are fixed relative to each other and the lifetime of the vibrational modes in the SAM of thiophenol are reduced as a result of the coupling with the metal on the SERS substrate. As a result of the uncertainty principle that holds an inverse relationship between the time and energy, the reduced lifetime of a vibration mode would obviously broaden the Raman linewidth.

Excitation Si peak (cm-1) Raman peaks of SERS peaks of wavelength thiophenol (cm-1) thiophenol (cm-1) (nm) centre width centre width centre width 457 520.7 4.5 1000.5 5.0 999.6 6.4 1025.0 5.8 1022.3 7.1 1092.0 14.1 1073.8 14.1 1583.9 14.9 1574.0 10.4 488 519.3 4.1 999.9 4.3 997.3 6.0 1024.6 4.9 1020.1 6.9 1091.9 11.2 1070.6 14.7 1583.5 14.3 1571.6 13.1 514 520.1 4.0 999.6 3.5 998.6 5.8 1024.2 4.3 1020.7 6.7 1091.4 10.2 1072.0 15.0 1582.8 11.3 1573.0 14.1 633 519.5 3.9 999.1 3.5 998.8 5.3 1023.7 4.3 1021.2 6.3 1091.0 9.7 1072.2 12.7 1582.2 15.1 1573.0 9.7 785 520.7 4.6 1003.7 4.1 1002.2 5.8 1028.2 4.8 1025.0 6.9 1095.6 9.8 1075.2 12.6 1586.9 16.2 1576.4 8.9

Table 6.3: Analysis of the wavelength dependence of Raman peaks of Si and thiophenol, and SERS peaks of thiophenol. The peak positions and the FWHM of peaks were determined with a Gaussian fit.

The peak positions of neat thiophenol and SERS thiophenol is quite consistent with the previously reported values by Joo et al. [231] within a value of ± 2 cm-1. However, it appears that 785 nm excitation may not be so well calibrated for the longer wavelength shift (cm-1) range of this instrument, as the peak positions are consistently higher by about 4 cm-1.

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6.3.2.2 Benzyl mercaptan

Benzyl mercaptan has the chemical formula of C6H5CH2SH (Figure 6.10) and is also known as benzylthiol, alpha-toluenethiol or benzenemethanethiol. A 10 mM solution of benzyl mercaptan was also used as an analyte for some of the SERS measurements in this thesis. The solution was prepared by diluting 119 μl of benzyl mercaptan (Sigma-Aldrich, ≥ 99.0%) in 100 ml of spectroscopic grade ethanol. A similar procedure was followed as discussed for thiophenol, to form a SAM layer of benzyl mercaptan on the OAD substrates prior to the investigation under the Renishaw inVia Raman spectroscope.

Table 6.4 shows the major vibrational modes assigned by [232, 233] for the SERS benzyl mercaptan spectrum and Figure 6.11 shows the experimentally observed SERS peaks of benzyl mercaptan adsorbed onto a Ag OAD film at 514 nm excitation.

SERS benzyl mercaptan (cm-1) Vibration assignment 648 C–S stretching and ring in-plane deformation 1000 Out-of-plane C–H wagging 1025 In-plane stretching C-H 1176 In-plane stretching C-H 1218 CH2 stretching 1580 In–plane stretching C-C 1599 Aromatic C–H in-plane bending and C–C ring stretching

Table 6.4: Experimental SERS peaks of benzyl mercaptan [232, 233].

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Figure 6.11: Peak positions in the SERS spectrum of benzyl mercaptan SAM excited with 514 nm.

6.3.3 SERS intensity dependence on the OAD angle

It has been reported that the maximum SERS signal for OAD substrates was achieved when the angle between the substrate normal and the deposition vapour flux is 86° [76, 86, 138]. In order to confirm the SERS signal dependence on deposition angle, a custom-made angle block (inset in Figure 6.12) with mounting positions from 78°-88° was mounted inside the deposition chamber to deposit films with a nominal thickness of 100 nm. The Ag OAD films were functionalised with a SAM of thiophenol before analysis under the Renishaw inVia Raman spectroscope. Thiophenol SERS spectra were recorded at 514 nm excitation with 50 objective for 10 s with averaging of three spectra. Five different locations of each sample were randomly chosen to study the signal variability across the sample. Figure 6.12 shows the intensity of the background corrected average thiophenol SERS spectra for different deposition angles. The background of the SERS spectra was removed using the penalised least squares method as discussed earlier (Section 3.3.4) and the spectra were further normalised to the laser power measured after the objective and the spectrum acquisition time.

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Figure 6.12: Intensity of average thiophenol SERS spectra for six different Ag OAD films with a nominal thickness of 100 nm deposited at angles of 78°-88° using a custom-made holder (inset).

In order to perform a comparison between the samples, the baseline-to-peak height of the four main thiophenol peaks located around 1000, 1021, 1072, 1573 cm-1 were calculated. The results are shown in Figure 6.13 representing the average peak intensity in y-axis together with standard deviation of the recorded spectra across the samples.

Figure 6.13: SERS intensity dependence on the vapour deposition angle. Page | 123

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The error in the deposition angle was estimated from the geometrical offset across the customised sample holder and positioning error in the deposition chamber. It was possibly bigger than  0.5°. As can be seen in Figure 6.13, the SERS signal tends to increase with increasing deposition angle, reaches a maximum at 86° and drops down at 88°. The variability of the SERS signal across the sample is significantly less for OAD 78° even as a relative error compared to the other deposition angles. It is interesting to note that with increasing deposition angle, the error bars also show a significant increase, confirming higher signal variability across the sample. It is understood that the SERS signal could be greatly enhanced when the molecules are located at hot spots (Figure 2.9). Therefore, the sample structures were further studied based on the SEM images of the samples using ImageJ software.

Figure 6.14: SEM images of Ag OAD films deposited at deposition angles of a) 78°, b) 80°, c) 82°, d) 84°, e) 86° and f) 88° with a nominal thickness of 100 nm.

Carefully controlling brightness and contrast thresholds of the SEM images of samples as consistently as possible, image analysis was performed on three separate slices of each image to calculate the average particle count and the percentage area of nanostructures. The errors associated with image margins when manually selecting the area of particles were unavoidable due to the non-perfect random shapes of nanoparticles.

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Sample ID Average particle count (per m2) % Area of NP OAD 78° 300 ± 30 48 ± 4 OAD 80° 415 ± 105 49 ± 4 OAD 82° 370 ± 65 54 ± 3 OAD 84° 720 ± 170 42 ± 4 OAD 86° 855 ± 70 40 ± 2 OAD 88° 635 ± 125 43 ± 3

Table 6.5: Image analysis results for Ag OAD samples fabricated using the customised holder for angles from 78° to 88°.

Interestingly, as can be seen in Table 6.5, the results obtained from the SEM image analysis suggest that the higher SERS signals (Figure 6.13) were received for the substrates with the higher average number of particles per unit area and lower percentage area of nanoparticles. Therefore, one can speculate that higher particle counts and smaller gaps in between particles may help to generate more hot spots. Consequently, the results support the conclusion that Ag OAD86° provides the best capability to produce high SERS signals and is consistent with the previous literature [76, 138].

6.3.4 SERS intensity dependence on the solid angle

A sample of Ag OAD86° 100 nm film functionalised with thiophenol SAM was used for the experiment. The 5, 20, 50L and 50 objectives were chosen for excitation of the sample with 514 nm wavelength and collected the backscattered SERS signal through the same objective. The thiophenol SERS spectra were collected from six different locations on the sample for each objective for 10 s with five accumulations to enhance the signal-to-noise ratio. The incident laser power onto the sample was controlled by the WiRE software and no signs of sample damage due to the chosen laser power levels were observed.

On one hand, one would expect to observe higher SERS signals with larger excitation area of the laser spot on the sample associated with the lower NA objectives compared to those of higher NA, provided that the excitation intensity is constant (power per unit area). The laser beam radius at the focal spot can be calculated [5, 222]:

 (6.3) s  0.61 . NA

However, the SERS signal that can reach the detector is limited to the collection cone of the light despite the excitation area and follows a linear relationship as can be seen in Figure 6.15. The average peak counts were normalised for the acquisition time and incident laser

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power onto the sample measured after each objective. The linear dependence holds a similar pattern to what was already observed for the Raman signal (Figure 6.1) and is consistent with previous work [210, 234, 235]. The assumption of a uniform distribution of the thiophenol SAM layer on metallic nanostructures appears justified for this analysis.

Figure 6.15: Average peak intensity of the four main thiophenol peaks plotted against the solid angle. The error bars represent the standard deviation of the measurements.

On the other hand, some loss of the SERS signal due to the coupling optics of Raman system is unavoidable even using a high numerical aperture objective. SERS is known to be an approximately isotropic process for the backscattering geometry and during the experiment, the SERS signal is only collected from one side. As an example, the use of a 0.95 NA objective, which is reported to be the maximum (https://micro.magnet.fsu.edu/primer /anatomy/numaperture.html) value that can be used in air, only enables a coupling efficiency of 34% of the total SERS signal to the detector, leading to a loss of more than a half of the SERS signal. As the interest of this thesis was to consider the near normal incidence of light onto the sample, the lowest numerical aperture lens 5 was used for most of the experimental SERS work.

6.3.5 SERS depth-of-field

In optics, the fundamental Gaussian transverse mode of TEM00 is generally used to describe the beam profile of most lasers [236]. The highest intensity region of the laser beam is known as the beam waist. To understand the SERS depth-of-field, 514 nm laser was focused Page | 126

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on a Ag OAD86° 100 nm sample with the help of the video camera using 5 lens. The shape of the beam profile observed through the laser-line-enabled camera view as described earlier in Section 6.2.3, was used as a visual guide to obtain the sharpest focus at the minimum waist of the beam, representing the focal plane when z=0. The z value of the motorised stage was automatically controlled in a series of measurements starting from  1000 μm to 1000 μm in 50 μm steps using the WiRE software. The incident laser power, number of accumulations of spectra and acquisition time were kept constant during the experiment. The variation of z at each step was responsible for the displacement of the sample surface from the focal plane in upward and downward directions. Figure 6.16 shows the strong dependence of the average peak intensity on the varying distance between the sample surface and the focal plane of the beam.

Figure 6.16: Average peak intensity plotted against the sample displacement from the focal plane of the laser beam.

The Lorentz fit shows the depth profile of the SERS field and half of the SERS signal is lost when the sample surface is 100 μm away from the beam waist, as can be seen from the FWHM. Presumably, the asymmetry in the data compared to the Si sample is due to optical aberrations when the laser beam was focused inside the transparent glass substrate [220]. Therefore, as seen for normal Raman scattering in Section 6.2.3, it is important to take care when focusing on the Ag OAD samples to avoid any variations in the SERS signal intensity that may arise due to sample surface displacements.

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6.3.6 SERS intensity dependence on the laser power

It is known from Equation (2.8) that the total intensity of the SERS signal is linearly dependent on the excitation laser power, provided that the SERS excitation cross section remains same. A sample of Ag OAD86° 100 nm functionalised with thiophenol was excited at 514 nm and used for experiment with the 5 lens. The laser power was controlled from 0.5%, 1%, 5% and 10% of total power in a set of six measurements for each power level. Applying high laser power level of 50% and 100% on the Ag OAD film during the initial measurements indicated burning on the samples; therefore, those power levels were not further used for the analysis. Figure 6.17 shows the excitation laser power dependence of the collected SERS signal counts normalised for the acquisition time of 10 s.

As can be seen, the average peak counts increase linearly with excitation laser power. The smaller error bars of the measurements show that higher repeatability can be achieved with lower excitation laser powers without any sample damage.

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6.4 Geometrical SERS experiments

Note: Parts of this Section have been modified from the following paper.

The outlined experiments in this section aims to provide a comparison between the near- side and far-side excitation of the SERS substrate. The excitation geometries are depicted in Figure 6.18.

Figure 6.18: Schematic of SERS signal collection geometry of a) near-side and b) far-side of the Ag OAD film deposited on transparent substrate.

The intensity ratio of far-side SERS signal to near-side SERS signal is referred to here as the geometrical enhancement factor (GEF), as shown in Equation (5.31). This indicates how much the SERS signal is enhanced when it collected through a transparent substrate compared to the normal collection geometry through air.

6.4.1 Streamline Raman mapping

A sample area of 400  400 μm2 was selected on a Ag OAD86° 100 nm film for Streamline Raman mapping as discussed in Section 3.3.4. The geometry of the nanostructures on the sample appeared to be islands, as can be seen in Figure 6.19 representing a section of the sample. The selected area on the sample covers the signal collection from a total of 400 points separated by 20 μm steps. The 5 lens, with a laser power of 1 mW was used for the mapping with the sample attached to the motorised stage. The beam spot diameter was

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around 5 μm. At each point the SERS spectrum was recorded for five seconds in static mode over the range of 784-1217 cm-1 covering the three main thiophenol peaks around 1000 cm- 1 (Figure 6.20). For the far-side mapping, the sample was carefully flipped over while it was attached to the customised sample holder to get the exact same area for the excitation. The accuracy of the excitation location was maintained using pre-defined sample margins and xy coordinates of the video viewer grid interactively. Note that the motorised stage of the Raman microscope records the position in the xy plane to 0.1 μm accuracy.

Figure 6.19: SEM image of the Ag OAD86° 100 nm film, revealing island-like nanostructures.

Figure 6.20: SERS counts of three main thiophenol peaks at 1000, 1021 and 1072 cm-1 recorded at a point on the selected area for the near-side during the Streamline mapping.

In order to understand the formation of hot spots on the sample, baseline-to-peak heights of the three main thiophenol peaks were calculated using the algorithm used by [237]. The Page | 130

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results of the analysis are shown in Figure 6.21 and Figure 6.22 for near-side and far-side respectively.

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It is interesting to observe the SERS counts variation across the sample which suggest that the hot spots are randomly distributed across the sample surface. In addition, as can been Page | 132

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in both Figure 6.21 and Figure 6.22, the lack of consistency in the peak heights could be also that different vibrational modes are enhanced more or less, depending on the orientation of molecules in a hot spot [238]. Consequently, the far-side to near-side signal ratio for each peak appears to be different. Nishijima et al. showed that the Raman-active vibrational modes of molecules experience different local field enhancements. The differences in effective density of states of surface plasmon modes at the Raman mode frequencies can be represented by the Purcell factor [39]. Nonetheless, the far-side SERS counts were always higher compared to the near-side counts for each vibrational mode (Note: the range of the colour scale is different for each of the mapped vibrational modes). The geometrical enhancement factor was found to range from 3.0-3.1, when the highest and the lowest peak counts for far-side and near-side were considered for each peak mapped in Figure 6.21 and Figure 6.22. For a better comparison, the total counts of all three peaks for the near-side and the far-side were compared as shown in Figure 6.23.

120

NS peak 1 NS peak 2 100 NS peak 3 NS total FS peak 1 80 FS peak 2 FS peak 3 FS total 60 Counts

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 -1 Peak counts (s )

The average geometrical enhancement factor (the ratio of the mean of far-side total counts to the mean of near side total counts) was found to be 2.3 and was significantly less than Page | 133

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the above quoted values. The variability in the SERS counts over a large sample area as seen on the Raman maps could be arising as the hot spots have a critical dependence on orientation with respect to the excitation source and polarisation distribution of molecules [11, 68]. In addition, the lack of perfect near-side and far-side alignment leads to uncorrected random patterns of enhancement and peak counts. The different analyte orientations create even more variability in SERS counts across the sample.

Mani et al. [239] studied the orientation of a SAM of thiophenol molecules adsorbed onto a silver surface, using the non-linear vibrational spectroscopy technique of sum-frequency generation (SFG). According to their discussion, several studies have shown that thiophenol molecules are either disordered or organised with the benzene ring (phenyl ring) slightly tilted, strongly tilted or orientated parallel to the substrate surface. Interestingly, the findings of their study support the fact that the thiophenol SAM layer is ordered on the Ag surface with the aromatic ring aligned perpendicular to the sample surface and the C-S bond tilted by ~37° from the sample normal. These results agree with the early SERS experimental work performed on mechanically polished and electrochemically roughened Au and Ag surfaces by Bryant and Pemberton with just a few degrees of difference in the tilting angle [240]. Later, Bryant et al. [241] also discussed the issues involved in achieving a good monolayer surface coverage of thiophenol molecules due to surface roughness in poly-crystalline Pt surfaces. The surface coverage appeared to be less for the poly- crystalline surface when compared with the single crystalline due to disturbances arising from the surface roughness leading to poor intermolecular cooperativity in the film.

Understanding the formation of hot spots is crucial when it comes to a real SERS substrate. As discussed earlier, most of the computational techniques have shown that the maximum plasmonic fields are observed in the interstitial spaces of closely packed nanostructures. Kleinman et al. did a comprehensive analysis of the contribution of hot spots to the SERS signal in the context of ensemble-averaged and single molecule conditions [11]. Therefore, the variations in signal intensity in the array of SERS hot spots in Ag OAD86° 100 nm are likely to be strongly dependent on the spatial density of nanostructures, polarisation dependence of formation of hot spots, the ratio and orientation of thiophenol analytes that are adsorbed in a hot spot compared to those adsorbed elsewhere. Nevertheless, a number of unanswered questions remained about the homogeneity of the SERS response of a sample and the formation of hot spots. A review article by Brown and Milton discussed the relationship between the SERS signal variability across various nanostructured samples and their enhancement factors and suggests that many hot spots contribute to nonuniform SERS enhancements across the samples [66]. Page | 134

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According to our current understanding, the contribution of thiophenol orientation effects in different Ag metal islands, the local symmetry of the thiophenol molecule and the proximity of a vibrational mode to a binding site are far too complex to interpret individually at each point of the Streamline Raman map of Ag OAD86° 100 nm sample. A broad discussion based on a computed, time-dependent density functional theory (TDDFT) for chemically adsorbed thiophenol on silver clusters is found elsewhere [242]. As an ongoing challenge for SERS researchers, achieving spatial uniformity may be better than higher enhancement factors for single molecule detection in terms of a reliable, reproducible SERS substrate.

6.4.2 Wavelength dependence

Prior to testing the wavelength dependence of the geometrically-enhanced SERS signal in terms of far-side to near-side SERS signal intensity, it was interesting to observe the wavelength-dependent Raman spectra of pure thiophenol. Figure 6.24 shows the average raw counts of pure thiophenol normalised to [0, 1] and the spectra are vertically offset for clarity.

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These results were used as a visual guide to observe any changes in the wavelength- dependent SERS thiophenol spectra. A summary of Raman peak positions and their peak widths was presented in Table 6.3. The peak around 1300 cm-1 was identified to be a contribution originating from the glass that is greatly enhanced at 785 nm but not at 514 nm as shown in Figure 6.25. Similar shaped backgrounds with a broad 1300 cm-1 peak have been reported by several groups for glass-supported SERS substrates [24, 243]. Smythe et al. also confirmed that this feature originated from the silica fibre and not from the thiophenol when SERS is detected at an optical fibre facet [24]. For 785 nm excitation, Kamemoto et al. [244] and Cui et al. [245] claimed a strong and broad fluorescence band for glass peaking around 1382 cm-1 and Tuschel [246] described it as a very strong and broad photoluminescence peak at 1400 cm-1.

Figure 6.25: Normalised counts of Raman spectra of glass at 514 nm and 785 nm excitation using OriginPro 9.1.

Meanwhile, in the recent review article by Yadav and Singh [247], a detailed discussion of all Raman-active peaks was presented for different oxide glasses, including borate, silicate, phosphate, borosilicate, borophosphate, aluminosilicate, and phosphosilicate. The chemical composition of standard microscope glass slides is generally known to be a mixture of SiO2,

Na2O, K2O, CaO, MgO, Al2O3, Fe2O3 and SO3. The three main peaks observed at 514 nm excitation can be assigned as Si–O–Si bending vibration in depolymerized structural units at 600 cm-1, Si–O–Si symmetric stretching at 790 cm-1 and Si–O–Si asymmetric stretching at 1100 cm-1 as quite commonly reported for silicate glasses [27, 247]. In addition, the broader Page | 136

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peak around 1300-1500 cm-1 at 785 nm is an indication of the presence of oxides in the form of either B2O3, P2O5 or Fe2O3 in silicate glasses [247]. However, why this peak is active only at 785 nm is not yet clearly explained [243], but is presumably a resonance Raman phenomenon due to coupling of the excitation to an electronic transition in one of the glass components [25, 248, 249].

Figure 6.26 shows the wavelength-dependent average thiophenol SERS spectra collected from Ag OAD86° 100 nm deposited on microscope glass with the far-side excitation geometry. The raw SERS counts have been normalised to [0, 1] and spectra are vertically offset for clarity.

The varying backgrounds of these spectra make it necessary to apply a background correction before calculating the average thiophenol peak intensities for geometrical enhancement factor calculation. The background of each SERS spectrum collected at excitation wavelengths of 457 nm, 488 nm, 514 nm, 633 nm and 785 nm was carefully removed using the adaptive penalised least squares method as mentioned earlier [144] and corrected for incident laser power and acquisition time. The baseline-to-peak heights of four main thiophenol peaks around 1000, 1021, 1072 and 1573 cm-1 were used for the average peak intensity calculation at each wavelength for both collection geometries. Figure 6.27 shows the average thiophenol peak intensities for ten different measurement Page | 137

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positions, plotted against the different excitation wavelengths for near-side and far-side illumination geometries.

It is interesting to see the significant increase in the average peak intensity with the increasing excitation wavelength. This increasing enhancement is surprising due to the peak plasmon resonance wavelength (Figure 4.23), which is expected to lead to a corresponding maximum in the SERS enhancement. These results can be due to chemical enhancement or surface-enhanced resonance Raman scattering (SERRS) of the adsorbed complex at longer wavelengths [79, 249-251]. The experimental geometrical enhancement factors for the different excitation wavelengths are shown in Figure 6.28. The highest GEF is seen at 488-514 nm excitation and the lowest at 633 nm. Remarkably, for all excitation wavelengths, the average GEF is greater than two and especially for the wavelengths of 488 nm and 514 nm it is peaking at more than 3. The LSPR maximum of the nanostructures (Figure 4.12) is seen around 500 nm and thus closer to 488-514 nm excitation compared to the other wavelengths.

The interaction of light with smaller nanoparticles highly depends on the complex dielectric function at the excitation wavelength and is related to the SERS enhancement [25]. From Mie theory, for a small spherical particle, the plasmon resonance condition is found when

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the real part of the metal dielectric function fulfils the requirement of r    2 medium , where medium is the electric function of the surrounding medium [43, 56, 61] as discussed in Section 2.3.1. For interest, Table 6.6 shows the wavelength-dependent complex dielectric function extracted from the uniaxial model for the five different excitation wavelengths.

Excitation wavelength Dielectric function Dielectric function (nm) (Extraordinary) (Ordinary) 457 -3.38 + 8.16i -4.41 + 5.79i 488 -0.57 + 11.2i -3.94 + 9.00i 514 2.70 + 11.8i -2.11 + 11.4i 633 8.67 + 7.75i 7.17 + 12.8i 785 9.80 + 3.61i 12.0 + 7.12i

Table 6.6: Wavelength-dependent dielectric function of Ag OAD86° 100 nm on glass extracted from the uniaxial model.

If the metallic layer was assumed to be represented by a spherical particle placed in air that has an effective uniaxial optical response, the plasmon resonance peak should occur when

r   2. As can be seen in Table 6.6, the condition is only met approximately at 514 nm and only for the ordinary direction. However, the broader width of the LSPR band in the absorption spectrum (Figure 4.12) suggests that there is a broad distribution of plasmon bands at different wavelengths due to the differences in particle sizes of randomly Page | 139

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distributed nanostructures and their interaction with neighbouring particles. Therefore, the above argument appears invalid as the effective medium is not equivalent to an ensemble of spheres.

The average GEF predicted by the extended Fresnel model (Figure 5.8) at the film-substrate boundary is in semi-quantitative agreement with the experimental results shown in Figure 6.28, except for the 785 nm excitation. A more quantitative comparison and thus a more rigorous test of the theory will require a suitable SERS substrate with reduced variability. This is a major challenge due to spot-to-spot SERS signal variation across nanostructured sample, in most of the commonly used SERS substrates [66]. It should be noted that the use of a finite collection aperture is unavoidable in the Renishaw inVia experimental set up. Therefore, extrapolating from finite (but small) NA to zero NA gives an increased GEF [20]. The higher geometrical enhancement factor measured at 785 nm, compared to the extended Fresnel model predicted value, could have arisen due to the interaction between substrate- induced photochemical effects and the charge transfer or resonance Raman effects as discussed earlier. Therefore, further work is recommended to carefully study the spectral changes of the SERS spectra against the excitation wavelength. Some useful discussion in this regard is found in a recently published review article by Yamamoto and Itoh [249], where significant changes in SERS spectra were identified to originate from a combination of electromagnetic effect, charge transfer effect and resonance effect, when the electromagnetic and charge transfer effects were no longer independent of each other.

6.4.3 Substrate material dependence

In order to understand the dependence of the geometrical enhancement factor on the substrate material, a 100 nm Ag layer was deposited on a transparent sapphire substrate at an angle of 86°, using oblique angle deposition as described earlier. Sapphire has a high index of refraction compared to the microscope glass (Figure 4.8). Figure 6.29 shows the SEM image of Ag OAD86° on sapphire with randomly distributed island-like structures. The Ag OAD86° 100 nm on sapphire sample was functionalised with thiophenol and was again used for SERS analysis with the five different wavelengths on the Renishaw Raman system. Figure 6.30 shows the corresponding average peak intensity for near-side and far- side signal collection geometries of the substrate. For comparison, the wavelength-dependent complex dielectric function extracted from the uniaxial model for the five different excitation wavelengths are shown in Table 6.7.

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Figure 6.29: SEM image of Ag OAD86° 100 nm deposited on a sapphire substrate (scale bar 100 nm).

Excitation wavelength Dielectric function Dielectric function (nm) (Extraordinary) (Ordinary) 457 -4.50 + 4.39i -4.46 + 3.83i 488 -5.51 + 7.18i -5.57 + 6.21i 514 -5.39 + 9.96i -6.11 + 8.75i 633 4.61 + 16.8i 1.86 + 17.8i 785 12.1 + 11.8i 11.7 + 14.6i

Table 6.7: Wavelength-dependent dielectric function of Ag OAD86° 100 nm on sapphire extracted from the uniaxial model.

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For all of the excitation wavelengths, the far-side SERS counts are higher than the near-side counts, confirming the geometrically-enhanced SERS signal (Figure 6.30). Unlike Ag OAD86° 100 nm on glass, the geometrical enhancement factor at 633 nm remains high for the sapphire substrate as shown in Figure 6.31 whereas it dropped to a lower value for glass. As discussed earlier, a red-shift in the maximum GEF was predicted by the extended Fresnel model (Figure 5.9) and agrees well with the experimental results.

Compared to the Ag OAD86° 100 nm on glass, the sample on the sapphire substrate appears to exhibit a high geometrically-enhanced SERS signal over a broader wavelength range of excitation. This will be useful for a number of potential SERS sensing applications in the UV and near IR spectral range. Overall, the maximum GEF is similar (within error) to glass and confirms a key prediction of the extended Fresnel model, compared to previous model of Jayawardhana et al. [20] as discussed in Section 5.4.

Interestingly, within the limitations of the measurement errors, the wavelength at which the GEF is a maximum, associated with a particular nanostructured substrate appears to have some correlation with the absorption spectra of the sample, as can be seen in Figure 6.32 from the average absorption spectra of Ag OAD86° 100 nm deposited on glass and sapphire. On the other hand, for Ag OAD86° 100 nm films on both glass and sapphire substrates, the maximum GEF is observed when the wavelength-dependent real part of the

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dielectric function changes its sign from negative to positive. Nonetheless, more reliable substrates with a wider range of dielectric base are needed to confirm the significance of this correlation. However, it should be noted that the corresponding effective plasma frequencies for both Ag OAD86° 100 nm samples are within a range (3.1-3.8)1015 s-1 which is significantly less than the plasma frequency of 141015 s-1 of bulk Ag [127, 252]. This red- shift can be explained due to increased surface scattering when the nano-void structures are made of dimensions less than the mean free path (52 nm) of the conduction electrons of Ag [59, 143].

Figure 6.32: Normalised averaged absorbance spectra of Ag OAD86° 100 nm deposited on glass and sapphire. The excitation laser wavelengths are marked by dashed lines.

6.4.4 Angle of incidence dependence

A silver OAD86° 100 nm film deposited on sapphire was used for this experiment with 633 nm excitation wavelength. The sapphire substrate was selected as the supporting substrate over the microscope glass due to its low background Raman and fluorescence spectra [244]. A manual rotation stage (Thorlabs, HFR007) was attached to the motorised stage of the Renishaw Raman system and was used to control the incidence angle of light onto the OAD substrate. Considering the working distance of the lens and the space occupied by the sample when rotating by 5° steps to achieve each angle of incidence, the 5 lens was selected for the excitation and collection of the backscattered SERS signal. Use of a

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lower NA lens prevented excessive spreading of incidence angles over the laser spot on the sample. Figure 6.33 shows a schematic of the angle of incidence on the sample in the near- side excitation geometry. The Ag OAD86° sample was rotated from 0-85° in 5° steps and the angle was measured from the surface normal. The thiophenol SERS spectra were recorded for both near-side and far-side excitation of the film.

Figure 6.33: Schematic of the near-side excitation geometry for the Ag OAD86° film with varying angles of incidence i .

Figure 6.34: Average peak intensity for near-side and far-side excitation plotted against the angle of incidence ranging from 0-85°. The error bars show the standard deviation of two measurements.

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As can be seen in Figure 6.34, the average peak intensity for far-side excitation is higher than for incidence angles ranging from 0-65°. The decrease observed in the far-side counts compared to the near-side for incidence angles from 70° to 80° may arise from the mismatch between the focal plane of the laser and the sample surface due to the higher degree of sample tilting and aberrations in the optical path [27]. No thiophenol SERS peaks could be observed above 80° for the far-side excitation geometry due to the noise in the spectrum. Notably, far-side intensity is more or less as expected, but near-side behaves differently to the extended theory (Figure 5.10). It is important to note that incident polarisation state of the laser and the collected light remains in a plane parallel to the laboratory coordinates (p polarised) and is independent of the angle of incidence.

A similar decrease in the SERS intensity with increasing angles of incidence was found in the literature [58, 218]. A study by Mubeen et al. experimentally showed that SERS spectra recorded from a nanoparticle-on-mirror substrate (i.e. similar to the near-side excitation geometry) is dependent on the incidence angle for p polarised light and is independent for s polarised light onto the sample [211]. For a Ag nanorod array with length ~870 nm fabricated by OAD, the maximum SERS intensity is observed when the incidence angle is 45°-50° relative to the sample surface normal [161]. Therefore, the dependence of SERS spectra on incidence angle is highly sensitive to the local field around the nanostructures and the polarisation state of the excitation source as experimentally shown by different research groups [253-257]. The rapid change in the far-side SERS intensity compared to the near-side with increasing incidence angles may arise due to the local change in the plasmon field around the nanostructures with respect to the polarisation direction. As polarisation state of the excitation laser was always parallel (p polarised) and remains unchanged, rotation of sample would obviously restrict the excitation of the neighbouring nanostructures that will be located just above or below the focal plane. The corresponding geometrical enhancement factor curve is shown in Figure 6.35 for incidence angles ranging from 0-80° with a cosine fit as a visual reference.

As can be seen in Figure 6.35, higher geometrical enhancement factors are observed closer to near normal incidence of the light onto the sample. The collection cone of the 5 lens is about 14° [234] and suggests that backscattered SERS signals occurred on the surface of the nanostructured layer have been efficiently collected at lower incidence angles. This represents the situation where the sample surface is almost in plane with the focal plane of the Gaussian laser beam.

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Nonetheless, the extended Fresnel model predicted GEF as shown in Figure 5.10 does not follow the trend of the experimental curve. While the experimental GEF decreases with increasing angle of incidence, the extended model predicts a GEF as high as 20 for an incidence angle of 85°. Notably, these angle-dependent experimental results contradict the higher far-side SERS enhancement reported by Viets and Hill for the angled tip of an optical fibre probe [18]. However, they follow the trend as previously reported by Jayawardhana et al. that the geometrical enhancement factor decreases as the NA of the objective increases [20]. The NAs used there were in the range of 0.12-0.85, whereas the NA of optical fibre probes would be expected to fall at the lower end of that range. Considering the error bars of the present experimental results, it seems likely that there would be no significant far-side geometrical enhancement in the SERS signal with increasing angles of end face in optical fibre probes.

However, it is important to highlight that the differences in the local surrounding of the laser spot on the sample and finite NA with the definition of the excitation angle of incidence remain significant in the experimental study compared to the theoretical prediction, where the metallic layer was approximated by an effective medium. The extended theoretical model also considers the refraction through each interface of the sample for the near-side and far-side, whereas the experimental angles of incidences were not corrected by applying the Snell’s law, considering SERS signal collection from Ag film-sapphire interface. Page | 146

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Additionally, as discussed in Section 6.2.3, confocal Raman microscopes work on the basis of a confocal pinhole setting to reject out-of-focus rays generated from the sample. Macdonald and Vaughan [258] and Everall [220] showed the different behaviours of opaque and transparent samples when examined through a confocal Raman microscope. They showed that the probability for an out-of-focus ray originating away from the tight focus of the laser, to be collimated and passed through the tight focus at the pin hole, is relatively small compared to the total number of Raman scattered rays at that point of the sample. This may also contribute to the far-side SERS signal compared to the near-side, when light transmitting in and out through the thicker transparent substrate, particularly at larger excitation angles of incidence due to the aberrations in the optical path. Therefore, considering the potential for systematic errors, the discrepancy between the theoretical prediction versus the experimental results need to be further investigated in future work, carefully considering reflection and transmission losses at each interface.

6.4.5 Polarisation dependence

In order to understand the variation in SERS signal counts with respect to the source polarisation direction, the sample was simply rotated about the surface normal by 90° for near-side and far-side measurements. Normally the SERS signal was collected when the source polarisation was parallel to the deposition vapour flux. This can be approximated as the direction parallel to the excitation of the extraordinary axis of the uniaxial model that was used to represent the effective optical constants of the Ag OAD86° 100 nm film. Rotating the sample by 90° (ordinary axis) may help to observe any polarisation dependent optical response of the scattered SERS signal due to the uniaxial anisotropy of the sample. A silver OAD86° 100 nm film deposited on glass was used for the analysis at 514 nm excitation wavelength. Figure 6.36 shows the average of 10 thiophenol spectra collected with near- side and far-side excitation geometry for the excitation of the extraordinary and ordinary axes of the sample. Spectra were not corrected to remove the background so that the slight differences in the shape of the spectra along two orthogonal directions could be clearly observed.

The experiment was repeated using benzyl mercaptan as the analyte and the corresponding spectra are shown in Figure 6.37. Background of the spectra has not been removed to highlight the variations in the SERS spectra for two the orthogonal orientations of the sample with respect to the direction of the source polarisation.

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Both Figures show the slight differences in the shape and intensity of the thiophenol and benzyl mercaptan SERS spectra in extraordinary and ordinary directions. These results suggest that the SERS signal is highly sensitive to the local changes occurring in the film and the orientation of the molecules with respect to the polarisation state of the laser source. It has been shown that the polarisation state of the local field that is experienced by the analyte molecule in a hot spot is very different to the polarisation state of the exciting beam [259].

Therefore, to understand the incident polarisation dependence on the SERS intensity of the sample with respect to the deposition vapour flux, an experiment was conducted to compare the SERS signal, with and without a polarisation filter inserted in the scattered beam path. The rotation angle was defined as zero when the incident light polarisation was parallel to the direction of the deposition vapour flux. A silver OAD86° 100 nm sample attached to a manual rotation stage was then rotated in the sample plane by 10° steps with respect to the source polarisation (fixed) in a complete circle of 0-360°. Due to the clearly observed differences between the benzyl mercaptan SERS spectra for incident polarisation in the extraordinary and ordinary axes, the Ag OAD86° 100 nm sample functionalised with 10 mM benzyl mercaptan solution was used for the analysis at 514 nm excitation. The baseline-to-peak heights of five SERS peaks at 648, 1000, 1026, 1176 and 1219 cm-1 were used for the average peak intensity calculation.

Firstly, the unpolarised SERS spectra were recorded followed by the polarised SERS spectra of benzyl mercaptan. For the polarised SERS analysis, a polarisation filter (PF) was inserted in the scattered beam path which acts as an analyser only to detect parallel polarised SERS signal (i.e. SERS signal polarised parallel to the incident beam polarisation) as per our experimental set up. Figure 6.38 shows the polar plot of average peak intensity of unpolarised and polarised SERS spectra corrected for incident laser power and acquisition time. The spectra were recorded for the near-side only due to the time constraints.

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As can be seen from Figure 6.38, random variations in the average peak counts are seen for the unpolarised and polarised SERS spectra at all rotation angles. No angular dependence of SERS intensity was observed, which contradicts Figure 6.37. It should be highlighted that Figure 6.37 shows an average of ten SERS spectra, whereas this analysis was limited to a single SERS spectrum for each rotation angle. Multiple measurements on the sample represent the SERS signal variability within the sample as represented by the error bars previously. Nonetheless, the reason for this contradiction was unclear at this stage, but could be due to the different chemical effects originating from non-totally symmetric vibrational modes of SERS spectra of multiple measurements over the sample [249]. Noticeably, similar to the scattered Si Raman signal (Section 6.2.4), the SERS signal is also polarised. It is recommended to continue the analysis with some other analytes, but this was beyond the scope of this thesis.

A study carried out by Farcau et al. [260] using a linear array of silver half-shells showed that the SERS scattered light was always polarised despite the polarisation direction of the excitation. Together with simulation results, they concluded that the polarisation of the SERS signal is fundamentally determined by the optical and structural properties of the nanostructures and is independent of the polarisation state of the excitation. However, the Page | 150

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work published by Jeong et al. [261] and Gkogkou et al. [262] claimed strong polarisation dependent enhancement on SERS spectra of aligned silver nanowire rafts and Ag nanoparticles on patterned Si substrates, respectively. Further, Jeong et al. [261] showed the disappearance of the polarisation dependence effect with disordered nanowires. For aligned nanowire rafts, the light polarised across nanowires exhibited 10-fold enhancement in SERS compared to the case of polarisation along nanowires.

Interestingly, a paper published by Zhao et al. [214] discussed the polarised SERS analysis of an array of Ag nanorods of length  870 nm fabricated by oblique angle deposition. Anisotropy was observed for both UV-Vis absorbance and SERS spectra of the sample. The experimental results support the observation that the maximum SERS intensity occurs when the light polarisation was perpendicular to the long axis of nanorods, as reported by Jeong et al. [261] previously. A full analysis of the SERS spectra of trans- 1,2- bis(4- pyridyl)- ethene (BPE) recorded at 10° rotation steps confirmed that the molecular orientation on the sample was not the cause for the observed anisotropy in the polarised SERS signal [214]. Therefore, it should be noted that the observed anisotropy in the SERS spectra is likely to represent the highly localised plasmon modes originated due to the strong interaction of electromagnetic coupling between nearest neighbouring nanostructures.

6.4.6 OAD layer thickness dependence

When investigating the geometrical enhancement factor, all of the work presented so far in this Chapter has used Ag OAD86° films with a nominal thickness of 100 nm as the model system. However, to understand the contribution of the thickness of the OAD layer to the geometrically-enhanced SERS signal, three samples were fabricated on glass substrates with a nominal thickness of 100 nm, 150 nm and 200 nm respectively. The corresponding SEM images of the samples are shown in Figure 6.39 a, b and c), and suggest that the nanostructures tend to increase in size and form clusters with increasing nominal thickness of the films. Figure 6.39 d) shows the distribution of particle diameter calculated using the SEM images based on the ImageJ software. The particle size distributions are shown by box plots where the median, first quartile and third quartile values are indicated on the plots, while the error bars signify the maximum and minimum. The nanostructured area coverage was 49.1%, 60.3% and 70.5% for Ag OAD films with the thickness in increasing order.

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The polarised absorption spectra measured along two orthogonal directions (parallel and perpendicular to the vapour flux) are shown in Figure 6.40. As can be seen from the Figure, the LSPR maximum is red-shifting with increasing film thickness. The difference in the plasmon resonance peaks seen along two orthogonal directions supports the fact that the films exhibit an in-plane anisotropy despite the variations in the film thickness and nanostructure size. The SEM image analysis of the films confirmed the variations in the size distribution of nanoparticles as can be seen by the boxplots in Figure 6.39 d).

The films were functionalised with 10 mM thiophenol for the SERS analysis with 514 nm excitation. The spectra were first collected when the excitation polarisation was parallel to the direction of the vapour flux (approximately the extraordinary axis) followed by the sample rotation by 90° to set the polarisation parallel to the ordinary axis. The background removed baseline-to-peak heights of the four main thiophenol peaks were used for the average peak intensity calculations for both near-side and far-side excitation. The average peak intensity plotted against the nominal film thickness is shown in Figure 6.41. Interestingly, for the near-side SERS intensity, the maximum of average (average of extraordinary and ordinary) peak counts of 3600 was found for 150 nm film compared to the values of 3400 and 2000 for 100 nm and 200 nm films, respectively. In contrast, the far- side SERS counts dropped significantly with increasing film thickness, with average counts of 9500 , 7900 and 4300 for Ag films of 100 nm, 150 nm and 200 nm, respectively. Page | 153

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Therefore, the corresponding average geometrical enhancement factors are 2.8, 2.2 and 2.2 for film thickness of 100 nm, 150 nm and 200 nm, respectively. The observed decrease in the GEF with increasing thickness can be explained as: if the analyte generating SERS is more uniformly distributed through the thicker films, the far-side suffers increasing attenuation when light passing through the absorbing Ag nanostructures whereas the near- side is less dramatically affected.

As discussed earlier in Section 4.6, in thicker nanocolumns or rods, the structures are generally inclined from the substrate normal towards the direction of the deposition vapour flux and due to the bundling effect the nanostructures appear to be closer spaced in the direction perpendicular to the deposition vapour flux. Therefore, higher signal counts observed for the ordinary excitation direction may be due to the excitation of more hot spots in the sample compared to the parallel extraordinary excitation. But, this is subject to uncertainty due to the error bars and relatively small differences between average intensities. Nevertheless, near-side extraordinary counts were higher than the ordinary counts only for the 100 nm film. No conclusion can be made based on the available results about what causes this difference when compared to the other OAD films and is out of the scope of the current thesis. Based on these results, thickness of the nanostructured Ag film might provide an additional parameter to test the extended theory. However, this has not been pursued here, as the variability in the SERS spectra appears to be relatively high to achieve a sufficiently sensitive test. In order to account for the contribution of the local Page | 154

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plasmon fields to the SERS spectra collected in two orthogonal directions, a carefully designed analysis is recommended with a precisely controlled nanostructured substrate with reduced spot-to-spot variation, rather than randomly distributed OAD film. However, to partially support the observed SERS results, an XPS analysis was carried out on the films to determine the surface chemical composition as discussed in the next Section.

6.5 XPS analysis of Ag OAD films

XPS was performed as a preliminary analysis to study the chemical composition of Ag OAD films and to confirm the formation of Ag-S bond. Prior to investigate the surface chemistry of the Ag OAD films, pre-cleaned control samples of glass, sapphire and Si were used to determine the surface chemical composition of themselves. The survey spectra were calibrated using the CHx component at 285.0 eV and Figure 6.42 shows the elements on each substrate.

Figure 6.42: The XPS survey spectra of uncoated a) glass, b) sapphire and c) Si substrates. Page | 155

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It is important to note that not all of the peaks in the XPS spectrum are from the ejection of electrons from the sample with the direct interaction of incident X-rays. Some of the unassigned peaks were identified to originate from the Auger peaks [147]. For example, the peak around 950-1000 eV characterises the energy of electrons ejected from oxygen atoms due to the filling of the K shell by an electron from the L shell that was coupled with an ejected electron from an L shell and referred as O KLL. The peaks of the elements found on each substrate, their binding energies and their atomic concentrations are shown in Table 6.8.

Substrate type Name Binding energy (eV) Atomic composition (%)

Glass O 1s 533 44.6 ± 0.3 Si 2p 104 26.8 ± 0.2 C 1s 285 22.6 ± 0.2 K 2p 307 3.0 Mg 2S 89 1.5 Ca 2p 348 1.3 Na 1s 1073 1.1 F 1s 686 0.2 Fe 2p 717 0.02 Sapphire O 1s 531 44.3 ± 0.2 Al 2p 75 38.0 ± 0.2 C 1s 285 16.6 ± 0.2 Ca 2p 348 0.4 S 2p 169 0.4 N 400 0.3 Silicon Si 2p 100 50.2 ± 0.4 O 1s 533 22.2 ± 0.3 B 1s 187 15.9 ± 0.2 C 1s 285 5.9 P 2p 134 5.8

As can be seen, the microscope glass is mainly composed of oxygen, silicon and carbon, whereas for sapphire the main elements are oxygen, aluminium and carbon. The ratios of O: Si: C and O: Al: C for glass and sapphire are 1: 0.6: 0.5 and 1: 0.9: 0.4 respectively. It was interesting to see the presence of carbon on all three substrates, but this is normal for all surfaces exposed to air and is referred to as adventitious carbon as discussed earlier in the thesis.

For the XPS analysis of Ag OAD samples, films deposited on glass, sapphire and Si substrates were used. The survey spectra and high-resolution scans of C, Ag and O were used for the

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comparison of Ag/C and O/C ratios. The samples functionalised with 10 mM thiophenol ethanolic solution were also used to confirm the formation of Ag-S bond. The samples were dried in N2 before loading into the vacuum chamber and high-resolution scans of C, Ag, O and S were used for the analysis. The main peaks of Ag OAD86° 100 nm film XPS survey spectra and the corresponding peaks when treated with thiophenol ethanolic solution are shown in Figure 6.43. As can be seen, the survey spectra are only assigned to the spectral lines of O 1s, Ag 3d, C 1s and S 2p.

The peaks found around 600 eV and 100 eV in Figure 6.43 a) and b) represent the binding energies for Ag 3p and Ag 4p respectively [263]. The Ag OAD86° 100 nm films deposited on glass, sapphire and Si in three different coating cycles were used to determine the Ag/C and O/C ratios (Table 6.9). The corresponding ratios of Ag/C for glass, sapphire and Si are 0.6, 0.7 and 0.7 whereas for O/C are 0.6, 0.4, 0.3 respectively. These results suggest that the deposition technique is reproducible in terms of having a reasonably equal silver to carbon atomic composition. Three scanned spots on each sample were used for the analysis with the assumption of homogeneous Ag coverage on the sample surface.

Based on the high-resolution scans of Ag 3d and O 1s, the presence of oxygen in the forms of AgO (367.4 eV) and Ag2O (367.8 eV, 529.5 eV) can be confirmed in addition to the elemental oxygen (531.0 eV) and silver (368.2 eV) [263]. Similar results have been reported for XPS spectra of Ag OAD films fitted with Gaussian–Lorentzian curves in the work published by [264] and the corresponding peak assignments can be found elsewhere [265- 267]. Therefore, it is clear that rapid oxidation of the silver surface when exposed to the Page | 157

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ambient environment under the experimental conditions may make a significant contribution to the SERS enhancement factors. However, the reason for the varying ratio from 0.6-0.3 of O/C for different supporting substrates needs to be further studied to make any firm conclusions. Nevertheless, the presence of carbon (284.6 eV) and carbon bonded to oxygen in the form of C=O (288.4 eV, 531.6 eV) and C-O (286.6 eV, 532.6 eV) suggest that amorphous carbon as well as some oxygen contamination was unavoidable even though the samples were stored in the desiccator immediately after the deposition.

The peak of S 2p that was centred on the binding energy of 163.0 eV [265, 268] can be used to confirm the presence of thiols on Ag OAD86° (Figure 6.43 b). A review paper published by Vericat et al. [269] lengthily discussed the chemical, thermal stability and quality of self- assembled monolayers of thiols and their challenges in technological applications. Interestingly, the authors confirmed that when dealing with SAMs attached to nanoscale materials, the structure and the charge distribution at the surface of the nanoparticles could induce different properties such as electron transportation and surface stress at the metal- S interface.

Table 6.9 shows a summary of the XPS analysis of Ag OAD86° films with varying nominal thickness of metal deposited on different substrates. For Ag OAD films deposited on glass and treated with thiophenol, the ratios of Ag/C, O/C and S/Ag are decreasing against the increase in the film thickness. It should be noted that most of the XPS signal is collected within a sampling depth of 10 nm and the probability of ejecting an electron that is deeper than 10 nm is very low due to the energy loss. On the other hand, with an increasing film thickness, the roughness of the films plays a significant role in the attachment of the SAM layer to the Ag surface due to different packing densities of the substrates [239]. At the same time, roughness may affect the calibration factors for elemental composition to reabsorption of photoelectrons arising from different depths in the 3D structure.

For thicker OAD films the XPS results may not truly represent the chemical composition of the sample especially the sulphur concentration. To understand the contribution of the increasing film thickness on the chemical composition of Ag OAD films, three OAD films with a nominal thickness of 100 nm, 150 nm and 200 nm deposited on Si substrates were used. Interestingly, the results suggest a gradual increase in the Ag/C ratio for increasing film thickness as one would expect when surface coverage of Ag becomes higher.

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Ag OAD86° sample ID Atomic composition (%) Relative ratios

C 1s Ag 3d O 1s S 2p Ag/C O/C S/Ag Deposited 100 nm 45.4 ± 29.0 ± 25.3 ± - 0.6 0.6 - on glass 0.5 0.4 0.5 100 nm + 34 ± 2 34.6 ± 25.5 ± 6.3 ± 1.0 0.8 0.2 thiophenol 0.7 0.5 0.4 150 nm + 36 ± 3 43 ± 2 17 ± 2 4.2 ± 1.2 0.5 0.1 thiophenol 0.4 200 nm + 57 ± 5 28 ± 4 14 ± 2 1.8 ± 0.5 0.2 0.06 thiophenol 0.9 Deposited 100 nm 47.9 ± 33.4 ± 18.7 ± - 0.7 0.4 - on 0.7 0.8 0.5 sapphire 100 nm + 41 ± 2 36.1 ± 21.7 ± 1.5 ± 0.9 0.5 0.04 thiophenol 0.2 0.5 0.8 Deposited 100 nm 51 ± 2 34.2 ± 15 ± 2 - 0.7 0.3 - on Si 0.9 150 nm 47.5 ± 39.8 ± 13.0 ± - 0.8 0.3 - 0.6 0.1 0.5 200 nm 47.1 ± 43 ± 1 9.6 ± - 0.9 0.2 - 0.6 0.4

Table 6.9: The atomic composition and the relative elemental ratios for Ag OAD86° films deposited on glass, sapphire and Si substrates.

A study by Kumar et al. confirmed that sample exposure to the ambient environment could lead to degradation of the LSPR due to the formation of non-plasmonic AgxO on the sample surface [266]. Furthermore, Yeo et al. showed that amorphous carbon gives a characteristic SERS spectrum, but it disappears when coated with a SAM of thiophenol [176]. Nonetheless, it should be highlighted that XPS results of this thesis are not quantitatively accurate enough to say there are significant problems with the quality of the layers. The SERS spectrum would show more analyte variability if that was the case, not just intensity variability. Therefore, to investigate the direct correlation between the surface chemistry and SERS intensity of nanostructured films, a series of well-controlled XPS experiments is highly recommended as future work. But unless interested in some other analytes, it should not be worth spending time as this study confirms the thiophenol spectrum is for an Ag-S bond. Nevertheless, it will be important to consider that formation of a “perfect” SAM layer on the nanostructured metallic layer is far-off from the reality.

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6.6 Conclusion

In this chapter, experimental results for both Si Raman and Ag OAD86° SERS confirmed their dependence on the key experimental factors of solid angle, excitation laser power, depth- of-field and source polarisation. A linear dependence of the solid angle and the excitation laser power on Raman and SERS was clearly shown. A tight sample focus is highly recommended for each objective when collecting the backscattered Raman/ SERS signal as the varying sample position against the focal plane of the objective resulted in dramatic variations in the Raman/ SERS intensity, especially for high NA objectives. Such signal variations may contribute to make any misleading conclusions about the repeatability and sensitivity of the SERS substrate, as well as the significance of differences in the geometrical enhancement factor.

The Streamline Raman maps of the near-side and far-side of a Ag OAD86° 100 nm sample confirmed that the far-side SERS counts are always higher compared to the near-side SERS counts for a larger scan area. The relative enhancement factors for different thiophenol Raman modes emphasised that the molecules experience different local field enhancements at different vibrational modes. Interestingly, the variation in the GEF across the sample and the reduced average GEF as can been seen from Raman maps, appeared to arise due to the adsorption effects of the analytes and the highly sensitive formation of hot spots in-between the randomly distributed nanoparticles.

The experimental results showed that the geometrically-enhanced SERS signal and hence the GEF was dependent on the excitation wavelength, angle of incidence and the Ag layer thickness of the SERS substrate. GEFs as high as 3.5 at 514 nm for glass OAD substrates and 3.2 at 633 nm for sapphire OAD substrates were experimentally observed. The GEF was wavelength dependent and the maximum was observed closer to the characteristic plasmon resonance peak of the nanostructured Ag substrate as predicted by the extended Fresnel model. The characteristic plasmon resonance peak of the nanostructured Ag substrate and the maximum GEF were red-shifted for the underlying transparent substrate of high refractive index sapphire. The increasing incidence angle with respect to the substrate normal when the polarisation of the source was kept unchanged showed a decrease in the GEF. The maximum GEF was found at the normal incidence of the light onto the sample. The wavelength-dependent and substrate material-dependent experimental results broadly agreed with the theoretical predictions of the extended Fresnel model, whereas the angle-dependent results were contradicting at larger angles of incidence.

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The polarised SERS signal analysis confirmed some anisotropy of coupling of the laser with the local plasmon field in SERS enhancement but was hidden when looking at the average peak heights. Increasing the metal layer thickness of the Ag OAD86° sample tended to decrease the GEF, but the maximum thickness that could be experimentally used to get geometrically-enhanced SERS signal is yet unknown. The XPS analysis suggested that the rapid oxidation of silver surface and carbon contamination when exposed to the ambient environment under the experimental conditions may contribute to variability in the SERS enhancement factors. The contamination was unavoidable even though the samples were stored in the desiccator immediately after the deposition.

The experimental work presented here was an effort to identify and compare the key parameters that contribute to the geometrically-enhanced SERS signal. Moreover, it should be highlighted that each of the above-discussed factors should be individually considered for a specific application such as designing an optical fibre probe with geometrically- enhanced SERS signal for sensing.

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7 Conclusion and Outlook

7.1 Thesis conclusions

The work presented in this thesis examines the far-side geometrically-enhanced SERS signal from a theoretical and experimental perspective. The results presented here were intended to identify and compare the key influencing factors that contribute to the geometrically-enhanced SERS signal as it had not been thoroughly studied in the literature until now. The main conclusions of the work are summarised below.

Nanostructured Ag thin films fabricated at an oblique angle of 86° with a nominal thickness of 100 nm were used as the SERS-active substrates in this thesis, as the fabrication method is cost effective and relatively reproducible compared to other advanced fabrication techniques. For oblique angle deposited Ag films, inter-sample reproducibility was relatively good and the spot-to-spot variability was about 10-15%. The in-plane absorption measurements and image analysis results together with standard ellipsometry measurements confirmed that these nanostructured Ag films are anisotropic due to the deposition technique, even when the particle height was less than 20 nm, and despite the visually uniform appearance of the island-like structures.

The observed anisotropy of the Ag OAD86° 100 nm film was studied in detail using Mueller matrix ellipsometry. The effective optical constants of Ag OAD86° 100 nm films fabricated on transparent dielectric substrates of microscope glass, sapphire and fused silica were analysed to compare the effect of the underlying substrate on the effective optical constants of the nanostructured films. The results show wavelength-dependent uniaxial anisotropy with the optical axis inclined towards the direction of the vapour flux. Furthermore, the effective uniaxial optical response of the island-like layers was highly sensitive to the refractive index of the underlying transparent substrate. The LSPR peak of the extinction coefficient in the uniaxial model was red-shifted with increasing substrate refractive index.

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Wavelength-dependent effective optical constants of Ag OAD86° 100 nm films deposited on glass and sapphire, as represented by complex numbers with a real refractive index and an imaginary extinction coefficient, were used to theoretically model the geometrically- enhanced SERS signal. A theoretical model based on complex Fresnel reflection and transmission coefficients was used to calculate the corresponding electric field intensities for the near-side and far-side excitation geometries, thus overcoming the absence of the absorbing metallic film in the initial Fresnel model proposed by Jayawardhana et al. [20]. The extended Fresnel model consistently showed an increased geometrical enhancement factor over the wavelength range of 300-1000 nm. The model predictions also show a red- shift in the geometrical enhancement factor curves with increasing refractive index of the underlying substrates, but do not support a significant increase in geometrical enhancement factor with increasing substrate refractive index. The maximum geometrical enhancement factors were blue-shifted compared to the LSPR maxima of the corresponding samples (Section 5.4).

The geometrically-enhanced SERS signal was experimentally studied primarily using Ag OAD86° 100 nm SERS active films with thiophenol as the test analyte. The Ag nanostructures deposited on glass and sapphire consistently showed far-side geometrical enhancement for all of the available excitation wavelengths, with the geometrical enhancement factor peaking for excitation wavelengths closer to the LSPR of the sample. These results were in broad agreement with the predictions of the extended Fresnel model. A significant red-shift in the maximum experimental geometrical enhancement factor was also observed for the high refractive index sapphire substrate. The experimental results confirmed that the geometrical enhancement factor for Ag OAD86° 100 nm films deposited on glass and sapphire were the same within the measurement errors, thus supporting the predictions of the extended Fresnel model. The simplified model proposed by Jayawardhana et al. [20] predicted that the sapphire substrate would outperform glass by 35%, but this was not supported by the observations presented here. The approximation that the geometrical enhancement originates close to the metal-dielectric boundary was justified by the exploratory nature of this experimental study, but could be considered as a future elaboration of the extended Fresnel model (see Section 7.2).

The dependence of the far-side geometrical enhancement on the angle of incidence of the excitation beam did not match the theoretical predictions of the extended Fresnel model, but this disagreement may be due to systematic errors considering the difficulty of focusing the laser onto the inclined surface. In general, further validation of the extended Fresnel model will require more uniform SERS substrates in order to reduce the measurement Page | 164

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errors. In addition, the geometrical enhancement factor in these Ag OAD86° samples appears to be highly sensitive to the hot spots located in the focal spot of the laser, as averaging over a large area reduced the corresponding geometrical enhancement factor. Increasing the nominal thickness of the Ag nanostructured layer tended to decrease the geometrical enhancement factor, presumably due to the increasing attenuation in the far- side SERS signal as it traverses the metal film. The difficulty in avoiding sample contamination under day-to-day laboratory conditions was clear from the XPS analysis with the presence of adventitious carbon and silver oxides.

7.2 Future work

The work presented in this thesis opens the glimpse of possible new research perspectives for further investigation.

The ellipsometry work included in this study was limited to randomly oriented nanostructured Ag samples fabricated by oblique angle deposition. Therefore, it is highly recommended to extend the work to some uniform and ordered SERS substrates with arrays of nanoparticles with high enhancement factors to determine their effective optical constants. State-of-the-art top-down manufacturing techniques would be required to achieve this level of control over the structure. This will enable to conceptually apply the extended Fresnel model to evaluate the geometrical enhancement factors at different wavelengths, which in turn may help to identify the optimal design of geometrically- enhanced-SERS substrates for future sensing applications. This is especially important for gold substrates which are less prone to surface contamination and are often functionalised with chemically-selective coatings for SERS sensing applications [2, 243, 269, 270].

Future ellipsometry work could be extended to carefully study the difference between the effective optical constants of thin nanostructured films measured in the near-side and far- side geometries. The observations in this thesis suggest that a carefully controlled experiment is essential for a more conclusive comparative study to draw any firm conclusions for the near-side and far-side effective optical constants of SERS substrates.

Moreover, the amorphous carbon contamination and oxidation of samples was unavoidable during their exposure to the laboratory environment. A study of the contribution of environmental factors to the effective optical constants of the sample based on ellipsometry would be beneficial to understand the variability in experimental geometrical enhancement

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factors due to practical time constraints that limit the possibility of immediate SERS analysis of samples after fabrication. This analysis would need to be supported by XPS analysis of the samples, as XPS provides complementary information to SERS and is sensitive over similar length scales.

It has already been shown in the literature that the SERS signal could be further enhanced when combined with thin film interference effects. Investigation of the effect of thickness of the dielectric substrate and the thickness of the nanostructured film for the optimised geometrically-enhanced SERS signal is recommended for future experimental work, potentially leading to as much as an order of magnitude additional enhancement on top of the well-known electromagnetic and chemical enhancement mechanisms. In addition, as suggested above, the increasing thickness of the nanostructured Ag layer that leads to a decrease in the geometrical enhancement factor appears to indicate that the height at which the scattering arises within the film should be taken into account in a more complete model.

In the SERS experiments included in this thesis, it was not straightforward to understand the contribution of hot spots to the overall geometrically-enhanced SERS, due to the randomly oriented nanoparticle shapes and gap distances. Precisely controlled and repeated patterns of engineered metallic structures can be used to understand the effect of these hot spots and localised fields to the far-side geometrically-enhanced SERS. However, it should be remembered that hot spots are notoriously variable and difficult to fabricate reproducibly. Other approaches will be needed to reduce the variability in the average SERS signal levels and this remains a major challenge for the entire field [4, 66, 67].

Considering the prospective applications of SERS in the fields of biomedicine and chemistry, to perform in-situ measurements and remote sensing with the use of optical fibre probes, one challenge is to transfer these nanostructured metal films to a facet of an optical fibre. The decal transfer technique [24] and hybrid-graphene based metal nanostructure fabrication [22] can be used to transfer arrays of metal nanostructures to the facet of the optical fibre. The former uses a thin polymer film to strip the nanostructures fabricated on a dielectric substrate. Nevertheless, non-optimal light coupling and excitation losses need to be further studied for the optimised geometrically-enhanced SERS signal in this scenario, due to the differences in the optical setup compared to the case reported in this thesis with metal films fabricated on thin dielectric substrates. On the other hand, the planar substrates considered here may find relevance for in-line SERS measurements through observation windows in chemical reaction vessels or pipelines.

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The combination of the theory-driven extended Fresnel model with experimental SERS results for the far-side geometrical enhancement provide a conceptual approach that can be implemented for many other SERS substrates, thus providing an opportunity for other researchers to study the potential of different substrates for a wider range of applications and with improved sensitivity. Overall, this thesis contributes to improve the current understanding of the subtle physics underlying the far-side geometrically-enhanced SERS signal.

Page | 167

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Author’s Publications and Presentations

[1]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Effective optical constants of anisotropic silver nanoparticle films with plasmonic properties”, Optics Letters, 2016. 41(23): p. 5495-5498.

[2]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Effective optical constants of anisotropic silver nanoparticle films with plasmonic properties: erratum”, Optics Letters, 2017. 42(6): p. 1092-1092.

[3]. M. N. M. N. Perera, W. E. K. Gibbs, S. Juodkazis, and P. R. Stoddart, “Wavelength and refractive index dependence of the geometrical enhancement in surface-enhanced Raman scattering”, Journal of Raman Spectroscopy, DOI: 10.1002/jrs.5190.

[4]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Surface- enhanced Raman scattering: effective optical constants for electric field modelling of nanostructured Ag films”, in Proc. SPIE 9921 Optics + Photonics (Plasmonics: Design, Materials, Fabrication, Characterisation, and Applications XIV) 2016, San Diego, USA.

[5]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, and P.R. Stoddart, “Modelling effective optical constants of nanostructured Ag films”, Australian and New Zealand Conference on Optics and Photonics, November 2015, Adelaide, Australia –poster presentation.

[6]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Surface- enhanced Raman scattering: effective optical constants for electric field modelling of nanostructured Ag films”, SPIE Optics + Photonics, August 2016, San Diego, USA – poster presentation.

[7]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “The wavelength dependence of the enhanced electric field in silver nano island plasmonic films”, IONS KOALA, November 2016, Melbourne, Australia – oral presentation.

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[8]. M.N.M.N. Perera, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Study of Far-Side Geometrical Enhancement in Surface-Enhanced Raman Scattering”, Swinburne Celebrates Research Conference, June 2017, Melbourne, Australia – poster presentation.

[9]. M.N.M.N. Perera, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Far-Side Geometrical Enhancement in Surface-Enhanced Raman Scattering with Ag Plasmonic Films”, Abstract submitted and an article in preparation for SPIE Nanophotonics Australasia Conference which will be held on December 2017.

[10]. M.N.M.N. Perera, D. Schmidt, W.E.K. Gibbs, S. Juodkazis, and P.R. Stoddart, “Understanding the dielectric substrate effect on effective optical constants of Ag plasmonic films”, Article in preparation for Applied Optics journal.

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