Ophthalmic Investigations Based on Multiphoton and Second Harmonic Generation Microscopy

這份論文所探討的是一系列利用多光子螢光暨倍頻顯微術(以下均統稱為多光子顯微術)所研究的眼科學問題，其中包含有：多光子影像解析眼球表面組織結構、多光子影像解析角膜病理組織、角膜屈光手術後以多光子影像追蹤其傷口癒合過程，及活體小鼠多光子角膜影像。較於傳統的病理學研究方法，利用光學切片的多光子顯微術，提供了一個低侵入性、低破壞性的手段，讓我們可以得到高解析度的組織影像。由於多光子顯微術係利用非線性光學光現象，因此在成像時，僅有顯微鏡物鏡焦點處具有足夠的光子量產生點狀的激發體積，因此，多光子顯微術在實用上具備有與共軛焦顯微鏡相同的高對比及空間辨析力。此外，由於在實作上，多光子顯微鏡多使用近紅外光雷射為光源，因此，它還有穿透樣品較深的特點，結合其穿透力較深以及點狀激發所帶來的三維空間辨析力，多光子顯微術，可以用於三維造影重建構，使我們對生物組織活動有更完整的了解。在生物組織中利用多光子顯微術成像的另一個好處就是無須外加標定，由於在細胞質中即有許多分子具有螢光的特性，加上細胞外間質的主要成份之一—膠原蛋白，會產生二倍頻訊號，因此，我們可以在不切片、染色的情況下，即得到細胞及細胞外間質交互作用的資訊，也因此，多光子顯微術不失為是個研究組織工程和傷口癒合的好方法。本論文著重於利用多光子顯微術研究眼科學，特別是角膜疾病相關的研究，最主要的原因就是借重多光子顯微術在無須額外染色即可得到膠原蛋白組織結構的特點。由於角膜對於可見光的穿透率高達90%以上，這樣的高度透明性，使得在傳統病理學上，我們很難用普通的光學方法直接檢查角膜，然而利用多光子顯微術可以無標定觀察膠原纖維結構的特性，我們可以突破傳統病理學檢驗法的瓶頸，加以多光子顯微術的對生物組織的低破壞性，未來極有可能將這項技術應用到臨床活體檢驗上。這本論文中，我們循序漸進的介紹了最初觀察豬眼及GFP小鼠的角膜的驗證性實驗，我們確立了多光子顯微術得用以揭露角膜中細胞及角膜基質的形態與排列，接著我們取得了感染性角膜炎以及圓錐角膜在多光子影像中的病理特徵，再接下來，我們將多光子影像應用於追蹤屈光手術後兔角膜的傷口癒合過程，最後我們更將多光子顯微術推進到活體小鼠角膜影像上，以期在不久的將來，能將這項技術推展為活體檢測的利器。This thesis demonstrates multiphoton applications in a series of ophthalmic investigations which include ex vivo ocular surface imaging, corneal pathology, monitoring post surgery wound healing and in vivo mouse cornea imaging. Compared to conventional histology, optical biopsy using multiphoton and second harmonic generation (SHG) microscopy provides a less invasive probing method in biological tissues. The confocal-like imaging quality and prolonged penetration in turbid media contribute to highly resolved, three dimensional imaging in biological tissues. In addition, cytoplasmic autofluorescence and SHG signals from biological crystalline structures provide label-free visualization of various biological tissues. For example, collagen, the most abundant molecule in mammals, is an effective second harmonic generator and allows imaging of biological structures to be achieved without extra labels. With this capability, it become feasible to dynamically observe the interaction of cells and extracecular matrices, tissue engineering and elucidating wound healing process. n this thesis, ophthalmic studies with multiphoton microscopy are particularly discussed due to the capability of label-free collagen visualization and potential of in vivo observation. In traditional ophthalmology examinations, it is difficult to observe stromal structure without biopsy, slicing and staining. However, with advances in modern optical microscopy, SHG imaging enables direct stromal imaging without traditional histology procedures which may cause undesired artifacts. The minimal invasiveness of multiphoton fluorescence and SHG imaging holds promise for clinically ophthalmic examination. n this thesis the effectiveness of multiphoton fluorescence and SHG microscopy in ophthalmic pathology has been proven by observing morphological changes in infectious keratitis and keratoconus. Moreover, time-lapsed investigation of post-refractive wound healing is qualitatively and quantitatively studied with the aid of multiphoton and SHG microscopy. In the end of the thesis, preliminary data of in vivo mouse corneal imaging is also demonstrated.Abstract I文摘要 IIist of Publications IIIable of Contents VIist of Figures IXhapter 1 Introduction 1hapter 2 Optical Biopsy in Ophthalmology Research 3.1. CONFOCAL MICROSCOPY 3.2. OPTICAL COHERENCE TOMOGRAPHY 4.3. MULTIPHOTON MICROSCOPY 4.3.1. The Absorption of Photons by Atoms[36] 5.3.2. Time-Dependent Perturbation Theory 6.3.3. Two-Photon Absorption Probability 7.4. SECOND HARMONIC GENERATION MICROSCOPY[34, 37] 9.4.1. Basic Principles 9.4.2. Interpreting SHG Images of Collagen Fibrils 12.4.3. The Advantages of Using SHG in Biomedical Imaging 14hapter 3 Anatomy of The Eye 15.1. GENERAL DIMENSIONS AND STRUCTURE OF THE HUMAN EYE 15.2. THE CORNEA 17.2.2. Corneal Epithelium 17.2.3. Bowman’s Layer (Anterior Limiting Lamina) 18.2.4. Corneal Stroma ( Substantia Propria) 18.2.5. Desçemet’s Membrane 18.2.6. Corneal Endothelium 19.3. THE SCLERA AND LIMBUS 19.4. THE UVEA 19.5. THE RETINA 20hapter 4 Materials and Methods 21.1. INSTRUMENTATION 21.1.1. The Homebuilt Multiphoton Microscope 21.1.2. Multiphoton Fluorescence and Forward/Backward SHG Microscope 22.1.3. Stage Scanning System 22.2. SAMPLE PREPARATION AND EXPERIMENTAL PROCEDURES 23.2.1. Ex Vivo Porcine Eye Imaging 23.2.2. Corneal Imaging of GFP mose 23.2.3. FWSHG and BWSHG on Ocular Surface 23.2.4. Corneal Pathology- Infectious Keratitis 24.2.5. Corneal Pathology- Keratoconus 24.2.6. Corneal Wound Healing- Conductive Keratoplasty 24.2.7. Corneal Wound Healing- Photorefractitve Keractomy 25.2.8. In Vivo Corneal Imaging 25.3. THE ANALYSIS ALGORISMS 26.3.1. Imaging Correlation Index (ICI) 26.3.2. Quantification of Corneal Infection 26.3.3. Keratocyte Density Calculation after PRK 27hapter 5 Proof of Principle 28.1. EX VIVO MULTIPHOTON IMAGING IN PORCINE AND GFP MOUSE EYES 28.1.1. Overview 28.1.2. Results and Discussion 28.2. FORWARD AND BACKWARD SHG IMAGING OF THE OCULAR SURFACE 32.2.1. Overview 32.2.2. Results and Discussion 33hapter 6 Corneal Pathology Revealed by Multiphoton Microscopy 38.1. INFECTIOUS KERATITIS[56] 38.1.1. Overview 38.1.2. Results and Discussion 39.2. KERATOCONUS [59] 43.2.1. Overview 43.2.2. Results and Discussion 44hapter 7 Corneal Wound Healing- Post-Surgical Multiphoton Imaging 50.1. INTRODUCTION 50.2. CONDUCTIVE KERATOPLASTY (CK)[60] 50.2.1. Overview 50.2.2. Results 51.2.3. Discussions 56.3. PHOTOREFRACTIVE KERACTOMY (PRK) 57.3.1. Overview 57.3.2. Results and Discussions 58.3.3. Conclusion 65hapter 8 In vivo Corneal Imaging 66.1. OVERVIEW 66.2. RESULTS AND DISCUSSIONS 66hapter 9 Conclusion 69hapter 10 Acknowledgments 71eferences 7