Development of a TEC Cryostage for Light Microscope and Its Application on Freezing Experiments of Microcapsules
Date Issued
2009
Date
2009
Author(s)
Chou, Shih-Chi
Abstract
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.
Subjects
Thermoelectric Cooler
Cryomicroscope
Intracellular Ice Formation
Microcapsule
Cryoprotectant
Type
thesis
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