光學顯微鏡熱電致冷低溫冷凍臺之研製並應用於微膠囊冷凍實驗

Abstract

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.