NEAR-INFRARED CONFOCAL RAMAN SPECTROSCOPY FOR REAL-TIME DIAGNOSIS OF CERVICAL PRECANCER SHIYAMALA DURAIPANDIAN NATIONAL UNIVERSITY OF SINGAPORE 2014 NEAR-INFRARED CONFOCAL RAMAN SPECTROSCOPY FOR REAL-TIME DIAGNOSIS OF CERVICAL PRECANCER SHIYAMALA DURAIPANDIAN 2014 NEAR-INFRARED CONFOCAL RAMAN SPECTROSCOPY FOR REAL-TIME DIAGNOSIS OF CERVICAL PRECANCER SHIYAMALA DURAIPANDIAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOMEDICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that this thesis is my original research work and has not been submitted anywhere for any degree in any university previously I have duly acknowledged all the sources of information which have been used in the thesis Shiyamala Duraipandian May 2014 Dedicated to my family and friends Acknowledgements It is indeed a great pleasure to thank those who helped me in the successful completion of my PhD First and foremost, I would like to express my hearty gratitude to my advisor Assoc Prof Zhiwei Huang for providing me an opportunity to pursue my PhD in his group I would want to thank my supervisor especially for his tutelage, indispensable technical advice and guidance, continuous and constant encouragement throughout my PhD journey from 2009 to 2013 I must express my special thanks for his prompt response to my queries and also his very immediate constructive comments in improving my manuscripts I would also like to acknowledge the clinical collaborators who are involved in this project, Assoc Prof Arunachalam Ilancheran, Assoc Prof Jeffrey Low Jen Hui and Dr Ng Soon Yau Joseph and also the nurses from the Department of Obstetrics and Gynaecology, National University Hospital, Singapore I would further like to thank my colleagues at Optical Bioimaging Laboratory: Dr Wei Zheng, Dr Mo Jianhua, Dr Lin Jian, Dr Lin Kan, Mr Teh Seng Khoon, Mr Wang Zi, Mr Wang Jianfeng and Mr Arnold for their help with my experiments On top of these, I would want to show my heartfelt appreciation to Mr Mads Sylvest Bergholt, one of my peer group members, for always being in corner as my best friend during my PhD journey and made it funfilled I would also like to express my solemn appreciation to my beloved parents and friends for their emotional support during my difficult moments Finally, I would like to acknowledge the funding agencies, the National Medical Research Council, and the Biomedical Research Council, Singapore I Publications (Peer-Reviewed Journals) S Duraipandian, W Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, "Near-infrared-excited confocal Raman spectroscopy advances in vivo diagnosis of cervical precancer," J Biomed Opt 18, 067007 (2013) S Duraipandian, W.Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, " Noninvasive analysis of hormonal variations and effect of postmenopausal vagifem treatment on women using in vivo high wavenumber confocal Raman spectroscopy," Analyst 138, 4120-4128 (2013) S Duraipandian, M S Bergholt, W Zheng, K Y Ho, M Teh, K G Yeoh, J B Yan So, and Z Huang, "Real-time Raman spectroscopy for in vivo, online gastric cancer diagnosis during clinical endoscopic examination," J Biomed Opt 17, 081418 (2012) S Duraipandian, W Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, "Simultaneous fingerprint and high-wavenumber confocal Raman spectroscopy enhances early detection of cervical precancer in vivo," Anal Chem 84, 5913-5919 (2012) S Duraipandian, W Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, "In vivo diagnosis of cervical precancer using Raman spectroscopy and genetic algorithm techniques," Analyst 136, 4328-4336 (2011) M S Bergholt, S Duraipandian, W Zheng, and Z Huang, "Multivariate reference technique for quantitative analysis of fiber-optic tissue Raman spectroscopy," Anal Chem 85(23), 11297-11303 (2013) II Publications (Conferences) S Duraipandian, M S Bergholt, W Zheng, and Z Huang, "Quantitative fiber-optic Raman spectroscopy for tissue Raman measurements," Proc SPIE 8939, (2014) S Duraipandian, W Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, "Integrated fingerprint and high wavenumber confocal Raman spectroscopy for in vivo diagnosis of cervical precancer," Proc SPIE 8572, (2013) S Duraipandian, W Zheng, J Ng, J J H Low, A Ilancheran, and Z Huang, "Effect of hormonal variation on in vivo high wavenumber Raman spectra improves cervical precancer detection," Proc SPIE 8214, (2012) S Duraipandian, W Zheng, and Z Huang, "Univariate and multivariate methods for chemical mapping of cervical cancer cells," Proc SPIE 8219, (2012) III Table of Contents ACKNOWLEDGEMENTS I PUBLICATIONS (PEER-REVIEWED JOURNALS) II PUBLICATIONS (CONFERENCES) III TABLE OF CONTENTS IV ABSTRACT VIII LIST OF FIGURES X LIST OF TABLES XVIII LIST OF ABBREVIATIONS XIX CHAPTER INTRODUCTION 1.1 Cervical cancer 1.1.1 Anatomy 1.1.2 Histology 1.1.3 Pathogenesis 1.2 Conventional screening procedures 1.3 Overview of Raman spectroscopy 1.3.1 The Raman effect 1.3.2 Raman instrumentation 1.3.2.1 Confocal Raman microscopy 1.3.2.2 Fiber-optic Raman spectroscopy 10 1.3.3 Excitation system 11 1.3.4 Spectrograph 12 1.3.5 Detection system 14 1.3.6 Fiber-optic probes 14 1.4 In vitro Raman spectroscopy for cancer diagnosis 19 1.5 In vivo Raman spectroscopy for cancer diagnosis 21 1.6 Data preprocessing 25 1.7 Multivariate statistical analysis 27 1.7.1 Principal component analysis 27 1.7.2 Classification algorithms 28 1.8 Biomolecular modeling 29 1.9 Thesis motivation and organization 31 1.9.1 Motivation 31 IV 1.9.2 Thesis organization 34 CHAPTER RAMAN SPECTROSCOPIC CHARACTERIZATION OF CERVICAL CELLS AND TISSUE SAMPLES 36 2.1 Introduction 36 2.2 Raman spectroscopy characterization of cells 38 2.2.1 Materials and methods 38 2.2.1.1 Cervix cell preparation 38 2.2.1.2 Micro-Raman instrumentation 38 2.2.1.3 Data preprocessing 39 2.2.1.4 Univariate and multivariate analysis 39 2.2.2 Results 40 2.3 Raman spectroscopy characterization of tissues 44 2.3.1 Materials and methods 44 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Gupta, "Depthsensitive Raman spectroscopy combined with optical coherence tomography for layered tissue analysis," J Biophotonics, 1-9 (2013) 168 .. .NEAR- INFRARED CONFOCAL RAMAN SPECTROSCOPY FOR REAL- TIME DIAGNOSIS OF CERVICAL PRECANCER SHIYAMALA DURAIPANDIAN 2014 NEAR- INFRARED CONFOCAL RAMAN SPECTROSCOPY FOR REAL- TIME DIAGNOSIS OF CERVICAL. .. adjunct to colposcopy for realizing real- time in vivo diagnosis of cervical precancer IX List of Figures Figure 1.1 Anatomy of cervix Figure 1.2 Stages of cervical precancer ... diagnostics 67 3.3 Real- time diagnosis 68 3.4 Conclusion 70 CHAPTER NEAR- INFRARED CONFOCAL RAMAN SPECTROSCOPY ADVANCES IN VIVO DIAGNOSIS OF CERVICAL PRECANCER