去在於果蠅(Drosophila)的行為研究上，發展出了多種行為觀測分析或者行為訓練的系統，為了研究不同的果蠅行為，這些觀測分析以及行為訓練系統各自有各自的用途以及特色，而近年來果蠅被視為研究睡眠對於晝夜週期行為之熱門對象。過去的睡眠實驗將果蠅置於試管內，中央使雷射光束偵測活動次數，然而在於分析上容易誤判為睡眠，且無法紀錄剝奪睡眠時，果蠅是否確實有受到干擾。本論文研發自動化即時分析果蠅睡眠平台與雷射追蹤干擾系統，使用即時影像處理分析，相較於過去研究利用雷射光束紀錄果蠅活動次數，解決睡眠誤判之問題，更能精確分析果蠅睡眠行為之特性，且此系統將整合雷射追跡系統，精準照射於果蠅身上，藉此觀察剝奪睡眠之效果，並將研究果蠅睡眠與學習記憶之間的關聯性，相信在未來對果蠅腦部的神經網路功能的了解必定能有大幅度的突破。
　　本論文之系統是由本實驗室過去所開發的”果蠅行為的即時影像分析與雷射刺激之系統”為基準進行改良及研發，首先設計一黑箱將系統完全罩住形成暗室，讓果蠅不受外界光源的干擾，藉由白光及紅外光LED發光模組模擬白天與夜晚之情境，紀錄並分析果蠅睡眠行為，並且改良果蠅行為觀測盒(Arena)之設計，能放置食物於內部以利果蠅長時間存活，設計多種食物放置方式來觀察果蠅的存活時間。相較於過去的系統，此系統可獲得更多且準確的數據(位置、速率、睡眠的次數及長度) ，並將延續過去即時追跡及分析果蠅行為搭配外在雷射刺激源，觀察之Arena數量增加至2個，可提升實驗效率，並能於暗室內利用紅外光雷射剝奪果蠅睡眠，分析不同雷射能量對於睡眠之影響程度，觀察行動力(Locomotion)表現及求偶行為(Courtship behavior)是否受到影響，提出最有效之剝奪果蠅睡眠的方法，使得日後研究人員在研究果蠅睡眠行為的實驗設計上擁有更大的自由度以及操作上更豐富的系統功能。

In the past, different kinds of analyzing or training systems have been developed for studying behavior of Drosophila melanogaster. In order to observe the different behaviors, these systems have their customized set of tools or special functions. Fruit flies have been used for decades in the study of sleep, and have become a popular model for the study of circadian behavior recently. The traditional method for monitoring fly activity involves counting the number of infrared beam crosses in individual glass tubes. However, if the fly moves away from the beam or if the fly sits right in the middle of it, too little or too much movement may be detected. Therefore, we provide a more faithful analysis system of locomotor activity and duration of sleep by using image processing and integrate with laser scanning module.
We present an “Automated Real-Time Analysis System with Laser Stimulated for Sleep Behavior Study of Drosophila”. The whole system is inside a black box; the flies need to be protected against the light from outside. An artificial light-dark environment of 12:12 (h) is considered standard by led module. Real-time analyze by using image processing, we can get the fly’s position and recognize the two different flies, and rotate the two-axis motor to make laser stimulations track the specific fly immediately. In addition, we design different kind of arena and observe which one is the best for flies to live. Our version not only provide the things which the previous system can do, but also can track the more detailed information (location, speed, duration and number of sleep bout) and provide more flexibility for experiments such as sleep deprivation and courtship behavior for researchers in the future.

致謝……………………………………………………………………………………I
摘要….....………………………………………………………………………….…II
Abstract……………………………………………………………...…………...…III
目錄……………………………………………………………………………….…IV
圖表目錄…………………………………………………………………………….VI
第一章　緒論 1
1.1 研究背景…..……………………………………………………………..…...1
1.2 文獻回顧……..…………………………………………………………..…...2
1.2.1 果蠅影像處理…………………………………………………………….2
1.2.2 果蠅學習與記憶平台…………………………………………………….3
1.2.3 果蠅睡眠平台…………………………………………………………….4
1.3研究動機 5
1.4論文計劃書架構 6
第二章　系統平台的架構與設計 23
2.1 硬體架構 (HARDWARE) 23
2.1.1分析系統模組 24
2.1.2 CCD Camera 24
2.1.3果蠅行為觀測盒與觀測盒置放機構 25
2.1.4白光與紅外光LED模組 26
2.1.5雷射掃描模組 26
2.2 軟體架構 (SOFTWARE) 27
2.2.1 LabVIEW 圖形化程式 27
2.2.2人機介面(GUI, Graphical User Interface) 27
2.2.3果蠅追蹤雷射訓練程式 28
第三章　實驗設計與結果 48
3.1實驗一 確認本系統是否能在暗室擷取果蠅之影像 48
3.2實驗二 觀察果蠅在果蠅觀測盒(ARENA)內的存活時間 48
3.3實驗三 研究果蠅日夜睡眠行為 49
3.4實驗四 研究不同IR雷射能量剝奪果蠅睡眠之程度 49
第四章　結論 61
第五章　未來工作 63
參考文獻 64

[1] Wolf, F.W. and Heberlein, U. Invertebrate models of drug abuse. J. Neurobiol. 54,
161–178 (2003).
[2] Guarnieri, D.J. & Heberlein, U. Drosophila melanogaster, a genetic model system
for alcohol research. Int. Rev. Neurobiol. 54, 199–228 (2003).
[3] Ho, K.S. & Sehgal, A. Drosophila melanogaster: an insect model for fundamental
studies of sleep. Methods Enzymol. 393, 772–793 (2005).
[4] Shaw, P., Ocorr, K., Bodmer, R. & Oldham, S. Drosophila aging. Exp. Gerontol. 43, 5–10 (2008).
[5] Konsolaki, M., Song, H.J., Dobbs, W. & Garza, D. P2–109 Drosophila models of Alzheimer’s-related pathways. Neurobiol. Aging 25, S255–S255 (2004).
[6] Jean-René Martin, Locomotor activity: a complex behavioral trait to unravel. Behavioural Processes. 64,145-160(2003)
[7] Jean-René Martin, A portrait of locomotor behaviour in Drosophila determined by a video-tracking paradigm. Behavioural Processes 67 207–219(2004).
[8] Dan Valente, Ilan Golani, Partha P. Mitra, Analysis of the Trajectory of Drosophila melanogaster in a Circular Open Field Arena. Plos One Issue 10(2007).
[9] Roseanna B. Ramazani, Harish R. Krishnan, Susan E. Bergeson, Nigel S. Atkinson, Computer automated movement detection for the analysis of behavior. Journal of Neuroscience Methods 162 171–179(2007).
[10] Heiko Dankert, Limimg Wang, Eric D Hoopfer, David J Anderson & Pietro Perona. Automated monitoring and analysis social behavior in Drosophila. Nature method 6, 297-208(2009).
[11] Kristin Branson, Alice A Robie, John Bender, Pietro Perona & Michael H Dickinson. High-throughput ethomics in large groups of Drosophila. Nature method(2009).
[12] Bjorn Brembs and Martin Heisenberg, Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator. The Journal of experimental biology 204 2849-2859(2001).
[13] Tyler A. Ofstad, Charles S. Zuker and Michael B. Reiser, Visual place learning in Drosophila melanogaster. Nature 474 204-207(2011).
[14] Hendricks, J.C. et al. Rest in Drosophila is a sleep-like state. Neuron 25, 129–138 (2000).
[15] Shaw, P.J., Cirelli, C., Greenspan, R.J. & Tononi, G. Correlates of sleep and waking in Drosophila melanogaster. Science 287, 1834–1837 (2000).
[16] Hendricks JC, et al. A non-circadian role for cAMP signaling and CREB activity in Drosophila rest homeostasis. Nat Neurosci. 1108–1115.(2001)
[17] Daniel Bushey, Kimberly A Hughes, Giulio Tononi and Chiara Cirelli, sleep, aging, and lifespan in Drosophila. BMC Neurosci.Vol.11,pp.56(2010)
[18] Ganguly-Fitzgerald, I., Donlea, J. & Shaw, P.J. Waking experience affects sleep need in Drosophila. Science 313, 1775–1781 (2006)
[19] Huber, R. et al. Sleep homeostasis in Drosophila melanogaster. Sleep 27, 628–639 (2004)
[20] Zimmerman, J.E., Raizen, D.M., Maycock, M.H., Maislin, G. & Pack, A.I. A video method to study Drosophila sleep. Sleep 31, 1587–1598 (2008)
[21]Giorgio F Gilestro, Video tracking and analysis of sleep in Drosophila melanogaster. Nature protocols vol.7 no.5(2012)
[22] Ulrich Stern, Edward Y. Zhu, Ruo He & Chung-Hui Yang. Long-duration animal tracking in difficult lighting conditions. Scientific Reports(2015)
[23] 吳建德, 研究果蠅行為的即時影像與雷射追跡系統之研發, 清華大學動力機械工程學系研究所碩士論文(2009)
[24] 劉宗和, 研究果蠅行為的即時影像分析與雷射刺激之系統研發, 清華大學動力機械工程學系研究所碩士論文(2010)
[25] Andrew M Leifer, Christopher Fang-Yen, Marc Gershow, Mark J Alkema and Aravinthan D T Samuel, Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nature Mrthod (2011)
[26] 齊振傑, 研究果蠅行為的即時影像分析與雙光雷射刺激之系統, 清華大學動力機械工程學系研究所碩士論文(2012)
[27]許智韋, 研發自動化即時分析與溫度調控果蠅社交行為之系統, 清華大學動力機械工程學系研究所碩士論文(2014)
[28] Joiner WJ, Crocker A, White BH, Sehgal A, Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441 757-760(2006)
[29] Pitman JL, McGill JJ, Keegan KP, Allada R., A dynamic role for the mushroom bodies in promoting sleep in Drosophila. Nature 441 753-756(2006)
[30] Crocker A, Sehgal A. Octopamine regulates sleep in Drosophila through protein kinase A-dependent mechanisms. The Journal of Neuroscience 28 9377-9385(2008)
[31] Wu M.-C, et al, Optogenetic control of selective neural activity in multiple freely moving Drosophila adults. PNAS (2014)