熱電致冷低溫顯微鏡系統模組化設計與微膠囊冷凍實驗

Abstract

本研究利用調整熱電致冷晶片的電流大小與方向即可控制表面溫度升降之特性，將其應用於低溫顯微鏡系統的冷凍台及冷卻系統改良設計，同時配合溫控電路與軟體程式達到升降溫之控制，最後並將整個系統模組化以增加使用上的便利性。改良之系統捨棄以往利用液態氮和加熱器來達到溫度控制的方式，並取代使用水浴循環機帶走廢熱的方法，以熱電致冷晶片設計完成小體積的冷卻系統，成功將系統總體積縮小，並可依實驗需求將冷凍臺替換為單層或三層熱電致冷晶片。改良後的低溫顯微鏡系統可達到之最低溫度為−68.6°C，最快降溫速率為 −120°C/min，恆溫控制溫度誤差均小於0.1°C，最大平均絕對誤差為0.0647°C。在本研究中亦以褐藻酸鈉為材料製作不同大小微膠囊以模擬人工細胞，並以低溫顯微鏡系統進行其低溫冷凍實驗，實驗結果顯示微膠囊會因為內外滲透壓不同而發生水分遷移現象，且當觸發囊壁外過冷水溶液時，囊壁外冰晶形成會隨即造成微膠囊發生細胞內凍結現象(IIF)。以不同濃度甘油改變微膠囊懸浮水溶液的濃度，將會隨水溶液濃度提高而降低水溶液的凍結點，因此也降低了微膠囊的IIF溫度。若使用乙醇為懸浮水溶液將囊壁外冰晶觸發IIF之因素剔除，微膠囊體積越小者，發生細胞內凍結之溫度越低，且體積接近之微膠囊，發生細胞內凍結的溫度範圍亦十分接近。另外，實驗結果亦顯示利用滲透壓特性令甘油或乙醇滲入囊壁內，可延緩囊壁外水溶液觸發結冰對於微膠囊發生細胞內凍結的時間與降低其IIF溫度，提高微膠囊內部的甘油或乙醇濃度，均可使得微膠囊發生IIF之溫度明顯降低。

In this study, we use the thermoelectric cooler (TEC) whose surface temperature can be controlled by adjusting the direction and magnitude of its current to improve the cold stage and cooling system of a cryomicroscope system. The temperature control circuit and the integrated software were also elaborated to make the cryomicroscope a modular system which is convenient to use. The original temperature control system using liquid nitrogen, heater and refrigerated circulation bath were replaced by the small-size TEC cold stage and cooling system. For different experimental demands, the TEC on the cold stage can be changed from single-stage TEC to tri–stage TEC. The lowest temperature achieved was −68.6°C while the highest cooling rate was −120°C/min. For the isothermal control of the cryomicroscope, the error of temperature was less than 0.1°C and the absolute mean error was 0.0647°C. Experiments were also performed to study the IIF behavior of microcapsules of various sizes by the cryomicroscope. It was observed that the microcapsules behaved as osmometers that water migrated across the cell wall when there was an osmotic potential. During freezing, IIF phenomenon of microcapsules occurred immediately after the ice front touched the microcapsule following the seeding of the supercooled solution. When the external solution was changed to glycerol solution of different concentrations, the IIF temperature decreased as the concentration of glycerol solution increased. When alcohol was used as external solution to preclude external ice formation during freezing, we observed that the smaller the microcapsule the lower the IIF temperature, and the variation of IIF temperature for microcapsules of the same size was small. When glycerol or alcohol was permeated into microcapsules due to the principle of osmolarity, the time when IIF occurred was delayed as well as the IIF temperature was lowered when the ice front touched microcapsules during freezing. Additionally, when we increased the concentrations of glycerol or alcohol concentration inside the microcapsules by permeation, the IIF temperatures of microcapsules decreased noticeably when the concentration increased.