本論文中，我們成功的將雷射光束掃描技術與鎖相放大器結合使用，開發出 一套高訊雜比與靈敏度的掃描技術。先利用老鼠尾巴富含的膠原蛋白作為樣品接 收二倍頻訊號，改變掃描的速度、像素面積、顯示大小等參數，找尋最適當的條 件。接著掃描骨骼肌細胞株。在不同的電脈衝刺激頻率下，肌肉收縮的頻率也隨 之改變，由分析所得的結果顯示，我們自行架設的系統可以測量動態的變化過 程，進行一維掃描時，每掃描一條線僅需要 5 ms，對於細胞的變化頻率 2 Hz 而 言，總共有100 的點去描述肌小節的動態變化過程，最後得到肌小節的變化頻率 與刺激頻率相同。同時也利用非線性光學影像的特性，掃描骨骼肌細胞的三維立 體影像圖，了解肌小節在細胞內的分布。最後掃描自發性收縮的肌胚胎心肌細 胞，觀察收縮與放鬆速度與長度的變化，並且比較加入腎上腺素後的影響。肌小 節收縮的速度在正常的情況下測得0.19 ± 0.11 μm/sec，放鬆的速度為 0.26 ± 0.13 μm/sec。然而進入腎上腺素後，收縮的速度加快為 0.33 ± 0.20 μm/sec (p = 0.029)，但是放鬆的速度為 0.26 ± 0.09 μm/sec 與正常的心肌細胞相似。長度百 分比變化量也試沒有太大的改變。
今後，我們會繼續使用此系統研究骨骼肌細胞株在不同電壓的刺激下以及改 變電壓脈衝的寬度，觀察對細胞的影響。此外還可加入其他的藥物，觀察對初代 心肌細胞的影響，在細胞層次下了解藥物的作用，做為動物實驗前的依據。不同 於其他技術與樣品，它可以簡單又快速的得到肌小節動態過程的變化。
第五章、參考文獻
1. Haupt, B.; Pelling, A.; Horton, M., Integrated confocal and scanning probe microscopy for biomedical research. The Scientific World Journal 2006, 6, 1609-1618.
2. Wu, Y.; Chen, H.; Chang, W.; Jhan, J.; Lin, H.; Liau, I., Quantitative assessment of hepatic fat of intact liver tissues with coherent anti-stokes Raman scattering microscopy. Analytical Chemistry 2009, 81, 1496-1504.
3. Brown, E.; McKee, T., Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nature Medicine 2003, 9, 796-800.
4. Huxley, A.; Taylor, R., Local activation of striated muscle fibres. The Journal of Physiology 1958, 144, 426-441.
5. Guo, Y.; Ho, P.; Savage, H.; Harris, D.; Sacks, P.; Schantz, S.; Liu, F.; Zhadin, N.; Alfano, R., Second-harmonic tomography of tissues. Optics Letters 1997, 22, 1323-1325.
6. Guo, B.; Guilford, W., The tail of myosin reduces actin filament velocity in the in vitro motility assay. Cell motility and the cytoskeleton 2004, 59, 264-272.
7. Ranvier, L., Du spectre produit par les muscles stries. Arch Physiol 1874, 6, 274-281.
8. Mann, D.; Urabe, Y.; Kent, R.; Vinciguerra, S.; Cooper, G., Cellular versus myocardial basis for the contractile dysfunction of hypertrophied myocardium.
Circulation Research 1991, 68, 402-415.
9. Yelamarty, R.; Moore, R.; Yu, F.; Elensky, M.; Semanchick, A.; Cheung, J., Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats. American Journal of Physiology- Cell Physiology 1992, 262, 980-990.
10. Lieber, R.; Loren, G.; Friden, J., In vivo measurement of human wrist extensor muscle sarcomere length changes. Journal of Neurophysiology 1994, 71, 874-881.
11. Powell, T.; Steen, E.; Twist, V.; Woolf, N., Surface characteristics of cells isolated from adult rat myocardium. Journal of Molecular and Cellular Cardiology 1978, 10, 287-290.
12. Nassar, R.; Reedy, M.; Anderson, P., Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte. Circulation Research 1987, 61, 465-483.
13. Roos, K.; Leung, A., Theoretical Fraunhofer light diffraction patterns calculated
from three-dimensional sarcomere arrays imaged from isolated cardiac cells at rest. Biophysical Journal 1987, 52, 329-341.
14. Roos, K.; Brady, A., Individual sarcomere length determination from isolated cardiac cells using high-resolution optical microscopy and digital image processing. Biophysical Journal 1982, 40, 233-244.
15. Kamgoue, A.; Ohayon, J.; Usson, Y.; Riou, L.; Tracqui, P., Quantification of cardiomyocyte contraction based on image correlation analysis. Cytometry Part A 2009, 75, 298-308.
16. Severs, N.; Slade, A.; Powell, T.; Twist, V.; Jones, G., Morphometric analysis of the isolated calcium-tolerant cardiac myocyte. Cell and Tissue Research 1985, 240, 159-168.
17. Rubart, M.; Pasumarthi, K.; Nakajima, H.; Soonpaa, M.; Nakajima, H.; Field, L., Physiological coupling of donor and host cardiomyocytes after cellular transplantation. Circulation Research 2003, 92, 1217-1224.
18. Van der Velden, J.; de Jong, J.; Owen, V.; Burton, P.; Stienen, G., Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes. Cardiovascular Research 2000, 46, 487-495.
19. Shevchuk, A.; Gorelik, J.; Harding, S.; Lab, M.; Klenerman, D.; Korchev, Y., Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.
Biophysical Journal 2001, 81, 1759-1764.
20. Stehle, R.; Kruger, M.; Pfitzer, G., Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid Ca2+ changes. Biophysical Journal 2002, 83, 2152-2161.
21. Telley, I.; Denoth, J.; Stussi, E.; Pfitzer, G.; Stehle, R., Half-sarcomere dynamics in myofibrils during activation and relaxation studied by tracking fluorescent markers. Biophysical Journal 2006, 90, 514-530.
22. Helmchen, F.; Denk, W., Deep tissue two-photon microscopy. Nature Methods 2005, 2, 932-940.
23. Franken, P.; Hill, A.; Peters, C.; Weinreich, G., Generation of optical harmonics.
Physical Review Letters 1961, 7, 118-119.
24. Gannaway, J.; Sheppard, C., Second-harmonic imaging in the scanning optical microscope. Optical and Quantum Electronics 1978, 10, 435-439.
25. Campagnola, P.; Millard, A.; Terasaki, M.; Hoppe, P.; Malone, C.; Mohler, W., Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophysical Journal 2002, 82, 493-508.
26. Perentes, J.; McKee, T.; Ley, C.; Mathiew, H.; Dawson, M.; Padera, T.; Munn, L.; Jain, R.; Boucher, Y., In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts. Nature Methods 2009, 6, 143-145.
27. Campagnola, P.; Loew, L., Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nature Biotechnology 2003, 21, 1356-1360.
28. Both, M.; Vogel, M.; Fink, R.; Uttenweiler, D., Second-harmonic generation imaging in muscle fibers. Confocal, Multiphoton, and Nonlinear Microscopic Imaging 2003.
29. Mohler, W.; Millard, A.; Campagnola, P., Second-harmonic generation imaging of endogenous structural proteins. Methods 2003, 29 (1), 97-109.
30. Plotnikov, S.; Millard, A.; Campagnola, P.; Mohler, W., Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres.
Biophysical Journal 2006, 90, 693-703.
31. Wallace, S.; Morrison, J.; Botting, K.; Kee, T., Second-harmonic generation and two-photon-excited autofluorescence microscopy of cardiomyocytes:
quantification of cell volume and myosin filaments. Journal of Biomedical Optics 2008, 13, 4018-4023.
32. Boulesteix, T.; Beaurepaire, E.; Sauviat, M.; Schanne-Klein, M., Second-harmonic microscopy of unstained living cardiac myocytes:
measurements of sarcomere length with 20-nm accuracy. Optics Letters 2004, 29, 2031-2033.
33. Plotnikov, S.; Kenny, A.; Walsh, S.; Zubrowski, B.; Joseph, C.; Scranton, V.;
Kuchel, G.; Dauser, D.; Xu, M.; Pilbeam, C., Measurement of muscle disease by quantitative second-harmonic generation imaging. Journal of Biomedical Optics 2008, 13, 4018-4028.
34. Both, M.; Vogel, M.; Friedrich, O.; von Wegner, F.; Kunsting, T.; Fink, R.;
Uttenweiler, D., Second harmonic imaging of intrinsic signals in muscle fibers in situ. Journal of Biomedical Optics 2004, 9, 882-892.
35. Zoumi, A.; Yeh, A.; Tromberg, B., Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.
Proceedings of the National Academy of Sciences of the United States of America 2002, 99, 11014-11019.
36. Zumbusch, A.; Holtom, G.; Xie, X., Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering. Physical Review Letters 1999, 82, 4142-4145.
37. Jhan, J.; Chang, W.; Chen, H.; Wu, M.; Lee, Y.; Chen, C.; Liau, I., Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing
structure-constituent correlation of tissues. Optics Express 2008, 16, 16431-16441.
38. Llewellyn, M.; Barretto, R.; Delp, S.; Schnitzer, M., Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans.
Nature 2008, 454, 784-788.
39. Hamburger, V.; Hamilton, H., A series of normal stages in the development of the chick embryo. Journal of Morphology 1951, 88, 49-92.
40. Akiyama, Y.; Furukawa, Y.; Morishima, K. In Controllable bio-microactuator powered by muscle cells, 2006; pp 6565-6568.
41. Suga, H.; Sagawa, K.; Shoukas, A., Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circulation Research 1973, 32, 314-322.
42. Song, L.; Wang, S.; Xiao, R.; Spurgeon, H.; Lakatta, E.; Cheng, H., β-Adrenergic stimulation synchronizes intracellular Ca2+ release during excitation-contraction coupling in cardiac myocytes. Circulation Research 2001, 88, 794-801.