Improvement of a TEC Cryomicroscope and Freezing Experiments on Zebrafish Embryos
Date Issued
2005
Date
2005
Author(s)
Wu, Chang-Hung
DOI
zh-TW
Abstract
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.
Subjects
低溫顯微鏡
冷凍保存
熱電致冷晶片
斑馬魚胚體
Cryomicroscope
Cryoprotection
TEC
Zebrafish embryos
Type
thesis