Improvement of a TEC Cryomicroscope and Freezing Experiments on Zebrafish Embryos

本研究的目的是改良前人所研製的熱電致冷低溫顯微鏡系統，對系統的效能進行加強，並以此系統進行觀察斑馬魚胚體發生細胞內凍結的情形。低溫顯微鏡系統包括冷凍台系統、顯微鏡與影像擷取系統、電路系統、數位類比訊號處理系統四個部份，在改良的部份主要是針對冷凍台的設計，將原有的夾合式固定座改用開放式盛裝低凝固點液體的玻片槽，解決水汽凝結影響觀察的問題，更簡化冷凍台的設計及減低實驗中樣本受損的機會。本研究的溫控以PID控制予以實現，藉由調整至合適的PID參數，使進行恆溫控制時，所得之最大超越量為0.45℃，而方均根誤差是0.34℃。並且將原本的降溫路徑漸近為弧線至目標溫度，使超越量減低，以測試結果來看，降溫速率在−100℃/min以內時，其超越量小於0.40℃。此低溫顯微鏡能夠達到的最低溫度與水浴槽的溫度有關，本研究中所到達的最低溫度為−49.58℃，而最快的降溫速率為−100℃/min。以此低溫顯微鏡系統進行了斑馬魚胚體的低溫冷凍實驗，了解斑馬魚胚體發生細胞內凍結的因子。實驗結果顯示了斑馬魚胚體外的冰晶形成與其成長的型態都會對胚體內發生凍結的機率造成影響，抗凍劑的使用也會將胚體發生凍結的溫度與機率降低，本研究使用DMSO以及甘油進行了實驗與比較。斑馬魚胚體早期(epiboly stage)或晚期(prim stage)，在−2℃時觸發使其周圍的水結冰都會100％造成IIF，在添加抗凍劑2M DMSO後，以觸發的方式使胚體外的溶液在較高的溫度形成冰晶，在其前端通過胚體不會隨即發生IIF，以此統計斑馬魚胚體由於溫度因子而造成細胞內凍結的溫度，早期的胚體為−18.65±5.14℃，晚期的為−21.06±6.82℃。以矽油代替胚體外的溶液，將胚體外冰晶形成所造成的影響完全除去，得到早期胚體在矽油中的IIF溫度為−20.98±1.55℃，而在浸泡過2M DMSO之後，使其降低至−33.33±2.59℃，晚期的則各為−19.60±1.57℃與−32.30±3.48℃。得知以矽油取代並除去胚體外溶液冰晶的形成以及添加抗凍劑對於降低斑馬魚的胚體內凍結溫度有明顯的效果。The objectives of this research are to improve the design and performance of a TEC cryomicroscope, and to perform experiments on the observation of IIF behavior of zebrafish embryos. The cryomicroscope system includes a cryostage, a microscope with image grabbing system, a current amplifying circuit, and A/D signal processing system. The improvement mainly focused on the design of the cryostage. By using a glass well containing low freezing point liquid such as ethylene glycol, the original design was simplified and the problem of moisture condensation was avoided. The temperature control was accomplished using PID control algorithm. Following tuning PID parameters, the accuracy was improved. For constant temperature control in the range from 10℃ to −35℃, the maximum error was 0.45℃, and root-mean-squared-error was 0.34℃. A method was also implemented to avoid temperature overshoot when cooling process is approaching an isothermal process. For a cooling rate of −100℃/min, the overshoot was less than 0.40℃. The lowest temperature that the cryomicroscope can reach was dependent on the temperature of the refrigerated circulation bath. The lowest temperature achieved in this research was −49.6℃ while the highest cooling rate was −100℃/min. The cryomicroscope was used to observe IIF behavior of zebrafish embryos. Experimental results showed extra-embryonic ice nucleation can initiate intra-embryonic ice formation of zebrafish embryos. The pattern of extra-embryonic ice nucleation affects the probability of IIF of zebrafish embryos. Loading of cryoprotectants depressed the temperature and probabilities of IIF for zebrafish embryos loaded with glycerol and DMSO were determined and compared. IIF temperatures of zebrafish embryos were significantly lower when immersed in silicon oil. For embryos loaded with 2M DMSO and immersed in silicon oil, the IIF temperatures were −33.33±2.59℃ and −32.30±3.48℃ for epiboly and prim stage zebrafish embryos, respectively.誌謝 i 摘要 ii ABSTRACT iii 目錄 iv 圖目錄 vi 表目錄 viii 第一章 前言與研究目的 1 第二章 文獻探討 3 2.1 低溫顯微鏡(Cryomicroscope) 3 2.1.1 簡介 3 2.1.2 對流式低溫顯微鏡 4 2.1.3 傳導式低溫顯微鏡 4 2.1.4 方向式低溫顯微鏡 4 2.1.5 熱電式低溫顯微鏡 6 2.1.6 綜論 6 2.2 熱電晶片( Thermoelectric Cooler, TEC) 9 2.2.1 簡介 9 2.2.2 特性 9 2.2.3 應用 12 2.3 PID控制法則 13 2.3.1 簡介 13 2.3.2 參數求法 16 2.4 斑馬魚(Zebrafish, Danio rerio) 21 2.4.1 簡介 21 2.4.2 低溫保存 21 第三章 研究設備與方法 23 3.1 實驗設備 23 3.1.1 冷凍台系統 24 3.1.2 顯微鏡與影像擷取系統 32 3.1.3 電路系統 34 3.1.4 數位類比訊號處理系統 36 3.1.5 系統整合 37 3.2 研究方法 40 3.2.1 溫度校正 40 3.2.2 參數調整 41 3.2.3 控制策略 41 3.3 樣本準備 46 第四章 結果與討論 47 4.1 溫度控制軟體 47 4.2 前置實驗 54 4.2.1 溫度校正驗證 54 4.2.2 PID參數的調整 54 4.2.3 降低超越量方法之參數決定實驗 58 4.3 溫度控制系統測試 65 4.3.1 不同速率降溫 65 4.3.2 不同目標溫度 69 4.4 斑馬魚胚體冷凍實驗 76 4.4.1 斑馬魚胚體冰晶觸發實驗 79 4.4.2 斑馬魚胚體脫水實驗 79 4.4.3 抗凍劑濃度的影響 85 4.4.4 溫度因子影響細胞內凍結 88 4.4.5 斑馬魚胚體與矽油冷凍實驗 92 第五章 結論與建議 96 5.1 結論 96 5.2 建議 99 參考文獻 10