造成不孕的原因當中有25 ~ 40 %是由於男性因素所引起(1)。造成男性不孕症的原因雖多但絕大部份是和精子的型態、數目及運動功能有關，因此提升男性精子品質與精子細胞之量測成為一個重要的事項。
    原子力顯微術具有原子級的解像能力，且不受限於導電材料與操作環境，可應用於各種生物的表面和微觀結構，故可作為奈米微晶結構的檢測工具。本實驗以原子力顯微術量測人類精子細胞形態構造，表面生物力學特性。從實驗的結果得知，精子細胞頭部高度、寬度和長度分別為0.96 ± 0.15 µm、3.53 ± 0.24 µm和4.79 ± 0.44 µm，而鞭毛的長度為35.94 ± 3.81 µm。表面的黏滯力約12.95 ± 1.23 nN，頂體抗體與精子細胞頂體鍵結力為441.67 ± 32.21 pN。藉由原子力顯微術分析精子細胞頭部奈米結構的差異變化，可以作為正常與異常精細胞的判別；利用原子力顯微術特有的量測力學特性來檢測頂體抗體與精子細胞頂體間專一性鍵結力之關係。

The semen quality was the major factor of infertility. Some clinical reports had also shown the correlation between the human sperm cell morphology and infertility. The objective of this study was investigated the ultrastructure of human sperm cell with atomic force microscope (AFM) which provided the examination of fine structural details of biomolecules already. The structure characterization of normal human sperm cells indicated that the height, width, length of the cell’s head were 0.96 ± 0.15 µm, 3.53 ± 0.24 µm, and 4.79 ± 0.44 µm respectively and the length of flagellum was 35.94 ± 3.81 µm. The mechanical property of the human sperm cell was obtained form the tip-cell adhesion, and the histograms and corresponding Gaussian distribution curves for adhesion force was estimated 12.95 ± 1.23 nN. Moreover, the novel interaction of force mapping between the tip with anti-acrosome modification and the acrosomal protein of the sperm cell was obtained from the AFM measurement. The specific binding force of normal human sperm cell was around 441.67 ± 32.21 pN. Compared of the nanostructure and the nanomechanical properties, we can regard as the discrimination of the normal and unusual human sperm cells.

目    錄
授權書
論文口試委員審議通過委員簽名處
中文摘要..................................................I
英文摘要................................................III
目錄......................................................V
附圖目錄...............................................VIII
附表目錄.................................................XI
第一章 緒論...............................................1
1- 1前言..................................................3
1-1-	1人類精子構造及功能..............................5
1-1-	2精子的生合成....................................7
1-1-	3人類生殖的過程.................................11
第二章材料與方法........................................13
2-1實驗原理.............................................13
2-1-1 原子力顯微技術....................................13
2-1-2 原子力顯微鏡基本架構..............................15
2-1-3 呈像原理及量測模式................................17
2-1-4 原子力顯微鏡之解析度..............................22
2-1-5原子力顯微鏡在生物學上之應用.......................23
2-1-6原子力顯微鏡在生物學應用上之優缺點.................24
2-1-7力－距離曲線.......................................27
2-1-8比例常數的量測.....................................30
2-2實驗製備.............................................33
2-2-1實驗藥品...........................................33
2-2-2實驗儀器...........................................34
2-3實驗步驟.............................................38
2-3-1物品配置...........................................38
2-3-2懸臂樑選用和彈性係數校正...........................39
2-3-3 探針修飾..........................................40
2-3-4作用力的量測.......................................45
2-4力學分析.............................................46
2-5 生物固定化技術......................................47
2-5-1 基材固定化........................................47
2-5-2 精子細胞固定化....................................47
第三章結果與討論........................................49
3-1精子細胞表面結構探討.................................49
3-2力學繪製.............................................57
3-2-1 表面力學繪製......................................57
第四章結論..............................................63
參考文獻................................................65
 
附圖目錄
圖 1-1 人類精子構造.......................................6
圖 1-2 人類精子細胞演化圖................................10
圖 2-1 原子力顯微鏡(AFM)的結構示意圖.....................15
圖 2-2 兩原子間的距離與相對位能的關係圖..................17
圖 2-3 接觸式(Contact mode)量測示意圖....................18
圖 2-4 非接觸式(Non-contact mode)........................19
圖 2-5 敲擊模式(Tapping mode)............................20
圖 2-6 為精子細胞的三維圖................................24
圖 2-7 力-距離曲線圖.....................................27
圖 2-8 探針壓剛體基質之力-距離曲線圖.....................31
圖 2-9 原子力顯微鏡(AFM)正面圖...........................34
圖 2-10 原子力顯微鏡(AFM)仰照圖..........................35
圖 2-11 CSC37型探針SEM影像圖（Micromash公司提供）........36
圖 2-12 CSC37型探針SEM結構圖.............................36
圖 2-13 生物鍵結步驟圖...................................41
圖 2-14 完成針尖修飾後的探針與載玻片.....................42
圖 2-15 針尖修飾後，光學顯微鏡下，探針之影像圖...........42
圖 2-16 針尖修飾後，螢光顯微鏡下，探針之影像圖...........42
圖 2-17 光學顯微鏡下，精子細胞之影像圖...................43
圖 2-18 螢光顯微鏡下，精子細胞之影像圖...................43
圖 2-19 在水溶液中操作原子力顯微鏡示意圖.................45
圖 2-20 量測黏滯力之力-距離曲線圖........................46
圖 3-1 為40 μm × 40 μm的精子細胞2D影像圖.................50
圖 3-2 為12 μm × 12 μm的精子細胞2D影像圖.................50
圖 3-3 為8 μm × 8 μm的精子細胞2D影像圖.... ..............50
圖 3-4 為20μm × 20μm的精子細胞3D影像圖...................51
圖 3-5 為15μm × 15μm的精子細胞3D影像圖...................52
圖 3-6 為10μm × 10μm的精子細胞3D影像圖...................52
圖 3-7 量測精子細胞頭部寬度之表示圖.... .................53
圖 3-8 為圖 3-7 所選取之區域高度分布圖...................53
圖 3-9 量測精子細胞頭部長度之表示圖............ .........54
圖 3-10 量測精子細胞鞭毛長度之表示圖.......... ..........54
圖 3-11框圈表示選取面積量測粗糙度之方式..................55
圖 3-12 室溫空氣下矽之黏滯力分布圖...... ................58
圖 3-13 AFM image........................................59
圖 3-14 精子細胞頭部表面黏滯力分布圖.....................60
圖 3-15 精子細胞添加頂體抗體之黏滯力分布圖...... ........58
圖 3-16 探針修飾抗體與精子細胞之作用力分布圖........ ....61

附表目錄.
表 1-1 精液檢查參考值.....................................4
表 2-1 CSC37型探針規格表.................................37
表 3-1 精子細胞之型態量測結果............................55
表 3-2 AFM所提供之統計資料表.............................56
表 3-3 為Silicon與Sperm cell之黏滯力比較表...............60
表 4-1 量測精子與文獻正常精子比較表......................63

1.Collins JA. Male infertility: the interpretation of the 
  diagnostic assessment. In: Mishell DR, Paulsen CA, Lobo 
  RA, eds. The Year Book of Infertility. Chicago: Year 
  Book Medical Publishers Inc 45, 1989.
2.Auger J, Kunstmann JM, Czyblik F, Jouannet P. Decline in 
  semen quality among fertile men in Paris during the past 
  20 years. N Engl J Med 332: 281-285, 1995.
3.Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. 
  Evidence for decreasing quality of semen during past 50 
  years. BMJ 305: 609-613, 1992
4.Cummins JM, Jequier AM, Kan R. Molecular biology of 
  human male infertility: links with aging, mitochondrial  
  genetics, and oxidative stress. Mol Reprod Dev 37: 345-
  362, 1994.
5.Schrader SM, Kanitz MH. In, Gold E, Schenker M, Lesley 
  B. State of the art reviews in occupational medicine, 
  reproductive hazards. PA, Hanley & Belfus, Inc., 405-
  414, 1994.
6.Bing, G. ; Quate, C. F. ; Gerber, C. H. phys. Rev. Lett. 
  1986, 56, 930
7.World Health Organization. WHO laboratory manual for 
  the   examination of human semen and sperm-cervical 
  mucus interaction. 3rd ed. Cambridge: Cambridge 
  University Press, 1992.
8.Breitbart, H. and Spungin, B. The biochemistry of the 
  acrosome reaction. Mol. Hum. Reprod., 3, 195–202. 1997.
9.Brucker, C. and Lipford, G.B. The human sperm acrosome 
  reaction: physiology and regulatory mechanisms. An 
  update. Hum. Reprod. Update, 1, 51–62. 1995
10.Binnig, G. ; Rohrer, H. ; Gerber, C. H. ; Weibel, E. 
   phys. Rev. Lett. 1982, 49, 57.
11.Gunning, A. P. ; Kirby, A. R. ; Morris, V. J. Imperial 
   College Press; October 1999, London, ISBN: 1860941990.
12.黃俊凱。台灣大學應用力學所93學年度碩士論文
13.曾文勝，林良平。科儀新知，1998, 19(6), 4-15.
14.Masanori yoshioka and Yukari mukai. “Immobilization of 
   ultra-thin layer of monoclonal antibody on glass 
   surface.” Journal of Chromatography, 556 (1991) 361-
   368.
15.Vinckier  Anja, Gervasoni Pietro, Zaugg Frank, Ziegler 
   Ursr, Lindner  Peter, Groscurth Peter, Pluckthun 
   Andreas, and Semenza Giorgio, “Atomic Force Microscopy 
   Detects Changes in the Interaction Forces between GroEl 
   and Substrate Proteins”, Biophysical Journal, 74, 3256-
   3263(1998). 
16.Ros Robert, Schwesinger Falk, Anselmetti Dario, Kubon 
   Martina, Schafer Rolf, “Antigen binding forces of 
   individually addressed single-chain Fv antibody 
   molecules ”Proc. Natl. Acad Sci. USA, 95, pp. 7402-
   7405, June 1998.
17.Joshi N, Medina H, Colasante C, Osuna A. 
   Ultrastructural investigation of human spermatozoon by 
   using atomic force microscope. Arch Androl , 2000; 44: 
   51-7
18.Joshi N, Medina H, Cruz I, Osuna J. 2001a. 
   Determination of the ultrastructural pathology of human 
   sperm by atomic force microscopy. Fertil Steril 75: 961-
   965.