Title Page

Abstract

Contents

Chapter 1. Introduction 15

1.1. Introduction to nonlinear optical techniques 15

Chapter 2. Theory of nonlinear optics 20

2.1. The wave equation for nonlinear optical media 20

2.1.1. Second-harmonic generation 23

2.2. Introduction to Raman scattering 25

2.2.1. Theory of Raman scattering 25

2.2.2. Raman spectroscopy 29

2.2.3. Molecular vibration 31

2.2.4. Surface-enhanced Raman scattering 33

2.2.5. Tip-enhanced Raman scattering 36

2.3. Theory of CARS 39

2.3.1. Fundamental of CARS 39

2.3.2. Intensity of CARS signal 45

2.3.3. Third-order nonlinear susceptibility 49

2.3.4. Phase matching condition of CARS 51

Chapter 3. Principles of multiplex CARS 56

3.1. Multiplex CARS utilizing a broadband source 56

3.1.1. Generation of a broadband Stokes source 59

3.2. MCARS experimental setup 61

Chapter 4. Classification of CARS spectral phase retrieval combined with principal component analysis 64

4.1. Introduction 64

4.2. Materials and methods 66

4.2.1. Materials 66

4.2.2. Contributions of the CARS response 67

4.2.3. Phase retrieval using the Kramers-Kronig relation 68

4.2.4. Coherent anti-Stokes Raman scattering spectroscopy 70

4.2.5. Spectral processing and data analysis 70

4.2.6. Principal component analysis 71

4.3. Results and discussion 74

4.3.1. Retrieved spectra of 4 marine oils 74

4.3.2. PCA processing of data set and classification of the samples 78

4.4. Conclusion 83

Chapter 5. Comparative analysis of lipids and collagen in skin tissues of cereblon ablated mice using a multimodal nonlinear optical system 84

5.1. Introduction 84

5.2. Materials and methods 86

5.2.1. Preparation of experimental animals 86

5.2.2. Preparation of skin sample and hematoxylin and eosin (H&E) staining 87

5.2.3. Materials 88

5.2.4. Multimodal nonlinear optical system 88

5.2.5. Spectral processing and data analysis 91

5.3. Results and discussion 92

5.3.1. Retrieved spectra of TAGs 92

5.3.2. Spectra of skin tissues of WT and CRBN KO mice 94

5.3.3. Quantitative analysis of lipid unsaturation in samples 96

5.3.4. Comparative analysis of collagen in WT and CRBN KO mice using SHG images 99

5.3.5. Characterization of intact biological tissues in their native conditions 103

5.4. Conclusion 105

Chapter 6. Summary 106

References 108

Curriculum Vitae 120

Table 2.1. Assignment of major Raman peaks in lipids 44

Figure 2.1. Energy level diagram of second harmonic generation. 24

Figure 2.2. Energy level diagram of the various scattering phenomena. 26

Figure 2.3. Vibrational modes of the molecule. 32

Figure 2.4. Surface enhanced Raman scattering generated by molecules adsorbed onto metal surfaces such as silver or gold nanoparticles. 35

Figure 2.5. Illustration of an AFM-based tip enhanced Raman effect. 38

Figure 2.6. Energy level diagram of CARS. 41

Figure 2.7. 3D structure of tristearin. 42

Figure 2.8. 3D structure of EPA in ethyl ester form. 43

Figure 2.9. Collinear phase matching diagram of (a) wave vector (b) beam geometry. 53

Figure 2.10. BOXCARS phase matching diagram of (a) wave vector (b) beam geometry. 54

Figure 2.11. Geometry of high numerical aperture phase matching using microscope. 55

Figure 3.1. Energy level diagram of multiplex CARS. 58

Figure 3.2. PCF used to generate a broadband source. 60

Figure 3.3. Scheme of experimental setup for MCARS. 62

Figure 3.4. The experimental setup of MCARS system. 63

Figure 4.1. Flow chart summarizing the process of performing phase retrieval from CARS measurements and applying PCA after baseline correction and normalization. 73

Figure 4.2. Extraction of phase retrieved spectrum of cod liver oil using raw CARS spectrum and NRB spectrum. (a) Raw CARS spectrum collected from cod liver oil. (b)... 75

Figure 4.3. Stacked plot of normalized retrieved spectra of anchovy oil (black), cod liver oil (red), salmon oil (blue) and krill oil (green) in the fingerprint region. 77

Figure 4.4. Loading spectra for the first three principal components showing the contribution to wavenumbers. 80

Figure 4.5. Scores plots to 2-D constructions of combinations of PC1, PC2 and PC3 marked by anchovy oil (black, triangles), cod liver oil (red, squares), salmon oil (blue,... 81

Figure 4.6. Scores plot to 3-D construction of PC1, PC2 and PC3 marked by anchovy oil (black, triangles), cod liver oil (red, squares), salmon oil (blue, stars) and krill oil (green, circles). 82

Figure 5.1. Scheme of experimental setup for a multimodal nonlinear optical system. 90

Figure 5.2. Stacked spectra of CARS spectral phase retrieval measured by five standard TAGs. 93

Figure 5.3. Ex vivo CARS spectral phase retrieval from WT and CRBN KO mice skin tissues. (a) Normalized spectra of average data from WT and CRBN KO mice skin tissues.... 95

Figure 5.4. Calibration curve for standard TAGs to quantify Nc＝c. 97

Figure 5.5. Calibration curve for standard TAGs to quantify Nc＝c/NCH2.[이미지참조] 98

Figure 5.6. SHG image of dermis from the skin tissue in the WT mouse. 100

Figure 5.7. SHG image of dermis from the skin tissue in the CRBN KO mouse. 101

Figure 5.8. Bar chart of relative SHG intensity measured from 150 frames of WT and CRBN KO, respectively. 102

Figure 5.9. qRT-PCR and western blot analysis of WT and CRBN KO mice. (a and b) The mRNA expression of collagen type I (a) and type III (b) was analyzed by qRT-PCR. The... 104