本研究應用本實驗室長期發展之聚氨基甲酸乙酯(polyurethane, PU)微型夾爪技術，建構出由壓電材料致動之微形夾持器系統，並增加一可移動培養皿放置平台，以模擬微粒子運動。在聚焦方面提出以非正規小波熵作為聚焦函數，由結果可看出不管是在定性或定量上本文所提出的聚焦函數性能(聚焦精度、靈敏度)皆優於其餘自動聚焦函數，且抗噪性佳。利用計算非正規小波熵低頻部份與後續的影像處理後再利用拉普拉斯法進行邊緣偵測，此方法有效的抑制雜訊並發揮拉普拉斯法優越的邊緣偵測能力，以此邊緣偵測方法與圓形霍式轉換實現PCSS法為主樣板比對法為輔之微粒子追蹤演算法，發展微粒子識別定位，成功對直徑 之多膠微粒子於水溫分別為 、 與 進行漂移運動時進行追蹤。最後結合本文所提出之聚焦函數與微粒子追蹤演算法成功的以單顆CCD實現三維空間中自動聚焦與影像伺服定位控制直徑 的微粒子傳輸與放置之應用實例，以便未來應用於生物醫學相關之微粒子操縱。

The present research is to implement a micromanipulating system by using a piezoelectronic-actuated micro gripper module, which applies the PU micro gripper technique developed by OME lab for a decade. A moveable platform for petri dish is added to simulate motion of particles. A focus function “Non-normalized Shannon Wavelet Entropy” is proposed to improve the performance over other focus functions on both focusing accuracy and sensitivity. In addition, the focus function has a good noise suppression performance. An edge detection method is improved though calculating the non-normalized Shannon wavelet entropy of low-frequency on image and using different image processing algorithms, and finally by employing mask of Laplacian to find the edge of image. The edge detection has a good noise suppression performance and retains fine edge detecting performance of Laplacian method. An algorithm of particles tracking based on PCSS and pattern matching is realized by utilizing both edge detection method and circular Hough Transform. The algorithm of particles tracking is successful in tracking microparticles with size around 3 , which exhibits drift motion in , and of water. Finally, tracking, transporting and manipulating of microparticles with size around 30~50 in 3-D workspace by using single CCD and employing the algorithm is implemented and tested for the future microparticles manipulation in biomedical research.

[1] F.C. Groen, I.T. Young, G. Ligthart, ”A comparison of different focus functions for use in autofocus algorithms,” Cytometry, vol. 6, no. 2, pp. 81–91, 1985.
[2] L. Firestone, K. Coo, K. Culp, N. Talsania, K. Preston, ”Comparison of autofocus methods for automated microscopy,” Cytometry, vol. 12, pp. 195–206, 1991.
[3] M. Subbarao, T.S. Choi, A. Nikzad, “Focusing techniques,” J Opt Eng, vol. 32, pp. 824–2836, 1993.
[4]T.T.E. Yeo, S.H. Ong, Jayasooriah, R. Sinniah, ”Autofocusing for tissue micro-scopy,” Image Vis Comput, vol. 11, pp. 629–639, 1993.
[5] A. Santos, C. Ortiz De Solorzano, J.J. Vaquero, J. M. Peña, N. Malpica, F. Del Pozo, “Evaluation of autofocus functions in molecular cytogenetic analysis,” Journal of Microscopy, vol. 188, pp. 264–272,1997.
[6]G. Yang, Bradley J. Nelson, ”Wavelet-based auto-focusing and unsupervised segmentation of microscopic images,” Proc IEEE/RSJ International Conference on Intelligent Robots and Systems, pp. 2143–2148, 2003.
[7]G. Yang, Bradley J. Nelson, ”Micromanipulation contact transition control by selective focusing and microforce control,” Proc IEEE International Conference on Robotics and Automation, pp. 3200–3206, 2003.
[8] H. Xie, W.B. Rong, L.N. Sun, “Construction and Evaluation of a Wavelet-Based Focus Measure for Microscopy Imaging,” Microscopy Research and Technique, vol. 70, pp. 987–995, 2007.
[9]T. Uemura, F. Yamamoto, F. Ohmi, “A high-speed algorithm of image analysis for real time measurement of two-dimensional velocity distribution,” Flow Visualization, ASME FED, vol. 85, pp. 129–34, 1989.
[10] K. Nishino, N. Kasagi, M. Hirata, “Three-dimensional particle tracking velocimetry based on automated digital image processing,”
J. Fluids Eng, vol. 111, pp. 384-391, 1989.
[11] K. Okamoto, Y. A. Hassan and W. D. Schmidl, “ New tracking algorithm for particle image velocimetry,” Experiments in Fluids, vol. 19, pp. 342-347, 1995.
[12] S.J. Baek, S.J. Lee, “A new two-frame particle tracking algorithm using match probability,” Experiments in Fluids, vol. 22, pp. 23-32, 1996.
[13] M. lshikawa, Y. Murai, A. Wada, M. lgchi, K. Okamoto, F. Yamamoto, “A novel algorithm for particle tracking velocimetry using the velocity gradient tensor,” Experiments in Fluids, vol. 29, pp. 519-531, 2000.
[14] X.D. Ruan, W.F. Zhao, “A novel particle tracking algorithm using polar coordinate system similarity,” Acta Mech Sinica, vol. 21, pp. 430-435, 2005.
[15] R.J. Adrian, “Particle-imaging techniques for experimental fluid mechanics,” Ann Rev Fluids Mesh, vol. 23, pp. 261-304, 1991.
[16]L.C. Gui, W. Merzkirch, “A method of tracking ensembles of particle images,” Experiments in Fluids, vol. 21, pp. 465-468, 1996.
[17] C.E. Willert, M. Charib, “Digital particle image velocimetry,” Experiments in Fluids, vol. 10, pp.181-193, 1991.
[18] H.B. Kim, S.J. Lee, “Performance improvement of two-frame particle tracking velocimetry using a hybrid adaptive scheme,” Meas. Sci. Technol., vol. 13, pp. 573-582, 2002.
[19] J. Willneff, “3D Particle Tracking Velocimetry based on image and object space information,” ISPRS Commission V Symposium, 2002.
[20]吳政達，「虛擬場景輔助進給與操縱遠端液體環境中之微粒子」，國立成功大學機械工程學系碩士論文，中華民國九十八年六月。
[21] F. Arai, “Micro tri-axial force sensor for 3D bio-micromanpulation,” International Conference on Robots and Automation, pp. 2744-2749, 1999.
[22] E. Vela, “Non-contact Mesoscale Manipulation Using Laser Induced Convection Flows,” International Conference on Intelligent Robots and Systems, IEEE/RSJ, pp. 913-918, 2008.
[23] H. Maruyama, “Immobilization of Individual cells by local photo polymerization on a chip,” Analyst, The Royal Society of Chemistry, vol. 3, pp. 304-310, 2005.
[24] T.P. Hunt, “Dielectrophoresis tweezers for single cell manipulation,” biomedical micro devices, Springer US, vol. 8, no. 3 , pp. 227-230, 2006.
[25] J.J. Gorman, “Probe-Based Micro-Scale Manipulation and Assembly Using Force Feedback,” Proceeding of the International Conference on Robotic and Remote System for Hazardous Environment, pp. 621-628, 2006.
[26]N. Yoshida, “Piezo-actuated Mouse Intracytoplasmic Sperm Injection (ICSI),” Nature Protocols, Nature, vol. 2, no.2, pp. 296-304, 2007.
[27]T. Trüper, “Transporting cells with mobile microrobots,” IEEE Proceedings Nanobiotechnology, IET, vol. 151, no. 4, pp. 145- 150, 2004.
[28]K. Han, “Fabrication of the Micro-gripper with a Force Sensor for Manipulating a Cell,” International Joint Conference, SICE-ICASE, pp.5833-5836, 2006.
[29]J. Park, “A Hybrid-type Micro-gripper With an Integrated Force Sensor,” Microsystem Technologies, Springer, vol. 9, pp. 511-519, 2003.
[30]O. Fuchiwaki, “Multi-axial micromanipulation organized by versatile micro robots and micro tweezers,” International Conference on Robotics and Automation, IEEE, pp. 893-898, 2008.
[31]N. Chronis, “Electrothermally activated SU-8 microgripper for single cell manipulation in solution,” Journal of Microelectromechanical Systems, IEEE, vol. 14, no. 4, pp. 857-863, 2005.
[32]F. Beyeler, “Design of a Micro-gripper and an ultrasonic manipulator for handling micron sized objects,” International Conference on Intelligent Robots and Systems, IEEE, pp.772-777, 2006.
[33]K. Kim, “Micronewton force-controlled manipulation of biomaterials using a monolithic MEMS microgripper with two-axis force feedback,” International Conference on Robotics and Automation, IEEE, pp. 3100-3105, 2008.
[34]S. Konishi, “Pneumatic Micro Hand and Miniaturized Parallel Link Robot for Micro Manipulation Robot System,” Proceedings of the 2006 IEEE International Conference on Robotics and Automation, IEEE, pp. 1034-1041, 2006.
[35]S. Lu, “Nanotube micro-opto-mechanical systems,” Nanotechnology, Institute of Physics, vol. 18, no. 6, 2007.
[36]B. Solano, A.J. Gallant, G.D. Greggains, D. Wood and M. Herbert, “Low voltage microgripper for single cell manipulation,”Advances in Science and Technology, vol 57, pp. 67-72, 2008.
[37]B.K. Chen, “Active Release of Micro Objects Using a MEMS Microgripper to Overcome Adhesion Forces,” Journal of Microelectromechanical Systems, IEEE, vol. 18, no. 3, pp.652-659, 2009.
[38]許志成，「形狀記憶合金驅動具力量感測器微夾爪之閉迴路控制」，國立成功大學機械工程學系碩士論文，中華民國九十八年六月。
[39]孫華偉，「影像伺服自動操縱液體環境中之微粒子」，國立成功大學機械工程學系碩士論文，中華民國九十九年六月。
[40]M. Subbarao, J.K. Tyan, “The Optimal Focus Measure for Passive Autofocusing and Depth-from-Focus,” Conference on Videometrics IV, SPIE, vol. 2598, pp.89-99, 1995.
[41]A. Adams, “The Camera,” New York Graphic Society, Boston, 1980.
[42]S.K. Nayar, Y. Nakagawa, “Shape from Focus,” Transactions on Pattern Analysis and Machine Intelligence, IEEE, vol. 4, no. 8, pp.824-831, 1994.
[43] N.K. Chern, P.A. Neow, M.H. Ang, “Practical Issues in Pixel-Based Auto-focusing for Machine Vision,” International Conference on Robotics and Automation, IEEE, vol. 3, pp. 2791-2796, 2001.
[44]J. Baina, J. Dublet, “Automatic Focus and Iris Control for Video Cameras,” Image Processing And Its Applications, IEEE, no. 410, pp.232-235, 1995.
[45] Matrox Electronic System Ltd. 2005. Matrox Inspector 8.0 user guide.
[46]R. Frigg, C. Werndl, “Entropy–A Guide for the Perplexed,” In Probabilities in Physics; Beisbart C. and Hartmann, S. Eds; Oxford University Press, Oxford, 2010.
[47]T. Jayasree, D. Devaraj, R. Sukanesh, “Classification of Transients using Wavelet Based Entropy and Radial Basis Neural Networks,” International Journal of Computer and Electrical Engineering, vol. 1, no. 5, 2009.
[48]R.C. Gonzales, R.E. Woods, Digital Image Processing, second edition, Prentice Hall, 2002
[49]J. Canny, “A Computational Approach to Edge Detection,” Transactions on Pattern Analysis and Machine Intelligence, IEEE, vol. PAMI-8, no. 6, pp. 679-698, 1986.
[50] I.E. Abdou, W.K. Pratt, “Quantitative Design and Evaluation of Enhancement/Thresholding Edge Detectors”, IEEE, vol.67, no. 5, pp. 753-763, 1979.
[51]P. V. C. Hough, “Method and means for recognizing complex patterns,” U. S. patent 3,069,654, 1962.
[52]R. O. Duda, P. E. Hart, “Use of the Hough Transformation to Detect Lines and Curves in Pictures,” Comm. ACM, vol. 15, pp. 11–15, 1972.
[53]N. Guil, J. Villalba, E. L. Zapata, “A Fast Hough Transform for Segment Detection,” IEEE Trans. Image Processing, vol. 4, no.11, pp.1541-1548, 1995.