林達德Lin, Ta-Te臺灣大學:生物產業機電工程學研究所周士棋Chou, Shih-ChiShih-ChiChou2010-05-052018-07-102010-05-052018-07-102009U0001-1508200900080600http://ntur.lib.ntu.edu.tw//handle/246246/180263低溫顯微鏡為觀察細胞於低溫環境的重要工具，藉由觀察結果可協助設計適用於特定細胞之冷凍保存方法。本研究所研製的冷凍臺是以熱電致冷晶片（Thermoelectric Cooler, TEC）為主體架構，藉由回饋控制方式以及設計的溫控方法，控制流經熱電致冷晶片的電流大小與方向，進而控制冷凍臺溫度。系統性能測試結果顯示，冷凍臺可控制之最低溫度為−50°C，降溫速率可達−80°C/min，且恆溫控制平均絕對誤差在0.1°C之內。所完成的低溫顯微鏡系統以微膠囊做為實驗觀察對象。在低溫環境下，當微膠囊外部水溶液的冰晶接觸到囊壁後，冰晶隨即觸發微膠囊產生細胞內凍結（Intracellular Ice Formation, IIF）的現象。本研究實驗設計是以微膠囊體積大小（0.1 ~ 0.4 mm）、降溫速率（−1 ~ −20°C/min）、囊內冷凍保護劑（二甲基亞颯（Dimethyl Sulphoxide, DMSO））濃度（1 ~ 3M）、囊外冰晶觸發影響等變因，進行各項冷凍實驗。在降溫速率與囊內DMSO濃度實驗中，得到在越慢的降溫速率與越高濃度的DMSO條件下，微膠囊能藉由脫水機制，降低發生細胞內凍結的機率。實驗結果顯示，置於1、2、3M DMSO溶液內的微膠囊，以−1°C/min降溫速率，由室溫至−50°C的過程中，發生細胞內凍結的機率依序是100%、4.8%、4.8%。另外，若以矽油取代微膠囊外部溶液，去除外部冰晶觸發和滲透壓影響，可更降低微膠囊內凍結的溫度，且體積越小的微膠囊，發生細胞內凍結現象的溫度越低。但在此項實驗中卻因排除脫水的機制，而最終導致所有的微膠囊發生細胞內凍結現象。當囊內為2M DMSO，體積0.1~0.2 mm、0.2~0.3 mm、0.3~0.4 mm大小的微膠囊，發生細胞內凍結的中間值溫度分別是−38.7°C、−30.4°C、−29.2°C。The cryomicroscope system is an important instrument for the observation of freezing behaviors of cells in low temperature environment. In this study, a cryomicroscope system was developed for transmission light microscopy based on TEC (Thermoelectric Cooler). The temperature of the cold stage is controlled by adjusting the magnitude and direction of the electric current supplied to the TEC using the feedback control algorithm developed in this research. For the TEC cryomicroscope system, the lowest temperature achievable is −50°C and the fastest cooling rate is −80°C/min. The absolute mean error of isothermal temperature control is less than 0.1°C. To test and verify the novel cryomicroscope system, we used microcapsules to simulate biological cells and observed their IIF (Intracellular Ice Formation) phenomenon. During freezing, IIF occurred immediately after the extracellular ice front in contact with the microcapsule membrane. Experiments were performed to investigate the factors affecting the IIF behavior. These factors include microcapsule volume size (0.1 to 0.4 mm), cooling rate (−1 to −20°C/min), intracellular DMSO (Dimethyl Sulphoxide) concentrations (1 to 3M), extracellular ice formation during freezing. For slower cooling rate and the higher DMSO concentration conditions, microcapsules can have lower probability of IIF due to dehydration. When microcapsules were suspended in 1, 2, 3M DMSO, cooled to −50°C at a cooling rate of −1°C/min, the probability of IIF were 100%, 4.8% and 4.8%, respectively. Additionally, when silicon oil was used as external solution to preclude external ice formation during freezing, the IIF temperatures of microcapsules were significantly lowered. The smaller volume of microcapsules resulted in lower IIF temperature. But IIF occurred for all of the microcapsules in the silicon oil because there was no dehydration during freezing process. When the intracellular solution of microcapsules was 2M DMSO, the median IIF temperatures of microcapsules, with 0.1~0.2 mm, 0.2~0.3 mm, 0.3~0.4 mm in diameter, were −38.7°C, −30.4°C and −29.2°C, respectively.誌謝 i要 iiBSTRACT ii錄 v目錄 viii目錄 xi一章 緒論 1.1前言 1.2研究目的 2二章 文獻探討 3.1低溫顯微鏡(Cryomicroscope) 3.1.1低溫顯微鏡簡介 3.1.2對流式(Convention)低溫顯微鏡 4.1.3傳導式(Conduction)低溫顯微鏡 5.1.4方向式(Directional)低溫顯微鏡 7.1.5熱電致冷式低溫顯微鏡 8.2熱電致冷晶片(Thermoelectric Cooler, TEC) 10.2.1熱電致冷晶片簡介 10.2.2熱電致冷晶片應用 14.3回饋控制理論 15.3.1 比例−積分−微分控制器簡介 15.3.2 比例−積分−微分控制器參數求法 17.4低溫保存(Cryopreservation) 19.4.1低溫保存簡介 19.4.2影響低溫保存之因子 21.4.3細胞內凍結 22.5微膠囊(Microcapsules) 23.5.1微膠囊簡介 23.5.2微膠囊包覆技術 24.5.3微膠囊在生物技術上的應用 26三章 研究設備與方法 28.1實驗設備 28.1.1冷凍臺系統 28.1.2散熱系統 31.1.3控制電路 32.1.4影像擷取系統 33.1.5溫控軟體 33.1.6系統架構 34.2研究方法 35.2.1溫度校正 35.2.2 比例−積分−微分控制器參數調整 37.2.3控制策略 41.3樣本準備 43.3.1實驗材料 43.3.2微膠囊製作方法 44.3.3掃描式電子顯微鏡樣本之製備與觀察 47.4實驗設計與規劃 48.4.1系統測試 48.4.2微膠囊細胞內凍結實驗 48.4.3微膠囊於觀測平臺上不同位置與細胞內凍結實驗 49.4.4體積大小與細胞內凍結實驗 49.4.5抗凍劑與微膠囊細胞內凍結實驗 49.4.6外部冰晶觸發微膠囊發生細胞內凍結實驗 50.4.7冷凍速率與微膠囊細胞內凍結實驗 51四章 結果與討論 52.1系統測試 52.1.1降溫速率 52.1.2恆溫控制 58.2微膠囊顯微構造圖 59.2.1微膠囊於光學顯微鏡呈像圖 59.2.2微膠囊於掃描式電子顯微鏡呈像圖 63.3微膠囊冷凍實驗 64.3.1微膠囊細胞內凍結實驗 64.3.2微膠囊於觀測平臺上不同位置之細胞內凍結實驗 64.3.3體積大小與細胞內凍結實驗 67.3.4抗凍劑與微膠囊細胞內凍結實驗 68.3.5外部冰晶觸發微膠囊發生細胞內凍結實驗 74.3.6冷凍速率與微膠囊細胞內凍結實驗 77五章 結論與建議 85.1結論 85.2建議 87考文獻 88application/pdf7977787 bytesapplication/pdfen-US熱電致冷晶片低溫顯微鏡細胞內凍結微膠囊冷凍保護劑Thermoelectric CoolerCryomicroscopeIntracellular Ice FormationMicrocapsuleCryoprotectantthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/180263/1/ntu-98-R96631031-1.pdf