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研究生： 蕭偉文
Hsiao, Wei-Wen
論文名稱： Biophysical Assessments on Biomaterials using Atomic Force Microscopy-Based Technology, in the Examples of Mouse Sperms and Astrocytes
指導教授： 張嘉升
Chang, Chia-Seng
潘榮隆
Pan, Rong-Long
口試委員: 張家靖
Chang, Chia-Ching
黃光裕
Huang, Kuang-Yuh
胡恩德
Hwu, En-Te
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2013
畢業學年度： 101
語文別： 英文
論文頁數： 123
中文關鍵詞： Atomic Force Microscopy 、Astigmatic Detection Microscopy 、Sperms 、Astrocytes 、Toxocara canis
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    • Cell biology has greatly assisted biologists in further understanding the biological mechanisms, which in turn are critical for researchers. Atomic Force Microscopy (AFM) is able to provide information regarding shape, height, surface roughness, volume, force, and elasticity as a platform for the multi-parameter analysis of cell functions.

    In this dissertation, we used AFM-based technology to study two different kinds of cells – sperms and astrocytes – to obtain information on morphological changes, force measurement, and cellular mechanics. First, we used AFM to measure the motility of a mouse sperm. Eukaryotic flagellum is responsible for the motile organelles that cause the migration of a mammalian sperm. The results showed that at a distance of about 18.5 μm from its head-tail junction, a lashing force of 0.96 ± 0.20 nN was measured. Its corresponding lashing torque was 1.77 ± 0.37 × 10-14 N•m. Our results reasonably concluded that the axonemal motility was linear dependent on the flagellum length of the sperm, and our developed measurement system could consistently determine the lashing force and torque of a sperm which might contribute to further studies concerning the mechanism of sperm transport and fertilization. Second, we used a combination of Astigmatic Detection Microscopy (ADM) and AFM techniques to study the pathogen-host interaction for astrocyte apoptosis triggered by Toxocara canis (T. canis) larval excretory/secretory antigen (Tc E/S), without tedious procedures and only relying on shape, surface structure, and height information. The variation in the pathology of a cell’s morphological changes was investigated with ADM and AFM analyses and then confirmed by Western Blotting. The results showed that the round cells increased as the concentration of Tc E/S antigen and incubated time increased, a similar trend. In addition, the mean height of apoptotic cells was approximately twice that of untreated normal cells, which meant there was correlation between the Tc E/S antigen treatment and cell height. For each cleaved caspase-3 in the cells co-cultured with Tc E/S antigen and incubated for 9 h, the corresponding intensities increased about 34-fold (34.4 ± 1.8) compared with those of the control cells. This method can provide researchers a perspective of understanding the limited information on the mechanism of astroglial injury and death during a T. canis larval invasion in a brain infection.

    Today, AFM is increasingly used in biological, biophysical and even biomedical research. As AFM instrumentation continues to develop, AFM measurements have boosted our understanding of various biological systems while revealing the promising potential in answering some unsolved questions in biology and in the establishment of novel biophysically analytical method.

    Acknowledgements 1 Abstract 3 Table of Contents 5 List of Figures 8 List of Tables 10 Abbreviations 11 CHAPTER 1: Prologue 13 1.1 Introduction 13 1.2 Scope of Dissertation 16 CHAPTER 2: Literature Review 19 2.1 The Fundamentals of Atomic Force Microscopy (AFM) 19 2.1.1 Working Principle 20 2.1.2 Operation Modes 21 2.1.3 AFM Probe: Cantilever and Tip 26 2.2 AFM in Cell Biology 29 2.2.1 Cell Morphology 31 2.2.2 Lateral Force Measurement 36 2.2.2 Cell Elasticity 39 2.3 Integration of AFM 42 2.4 High-Speed AFM 48 2.5 Intracellular Imaging of AFM 51 CHAPTER 3: Motility Measurement of a Mouse Sperm by Atomic Force Microscopy 53 3.1 Introduction 53 3.2 Materials and Methods 57 3.2.1 Preparation of Mouse Sperm 57 3.2.2 Modification of AFM Cantilever Tip 58 3.2.3 Force Measurement of Sperm 61 3.2.4 Analysis of Force Measurment and Toque Calculation 63 3.3 Results 65 3.4 Discussion 71 CHAPTER 4: Biophyscial Analysis of Astrocytes Apoptosis Triggered by Larval E/S Antigen from Cerebral Toxocarosis-Casuing Pathogen Toxocara canis 78 4.1 Introduction 78 4.2 Materials and Methods 83 4.2.1 Preparation of Larval ES Antigen 83 4.2.2 Co-culture of Astrocytes with Tc E/S Antigen 85 4.2.3 Cleaved Capase-3 Assessed by Western Blotting 86 4.2.4 Astigmatic Detection Microscopy (ADM) Imaging 87 4.2.5 Height Measurement Using AFM 90 4.2.5 Statistical Assessment 91 4.3 Results 92 4.4 Discussion 105 CHAPTER 5: Epilogue 108 References 110

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