• 한국가축번식학회지
• /
• 제25권4호
• /
• pp.371-379
• /
• 2001

본 연구는 체외에서 생산된 소 수정란의 동결을 위한 최적치 조건을 규명할 목적으로 실시하였다. 동결을 위하여 체외에서 생산된 8 세포기, 상실배기 및 비반포기 단계의 수정란을 공시하여 EC 5.5 동결온액에 20초 동안 노출시키고, 각 용기에 장착한 후, 즉시 -196$^{\circ}C$ 액체질소에 침지하는 유리화동결법을 채택하였다. 그 후 0.5 M, 0.25 M 및 0.121 M sucrose 용액에서 각 1분간씩, 연속으로 응해 한 다음, 10 % FBS가 첨가된 CR Iaa 배양액으로 옮겨 배양하였다. 그 결과 수정란의 재팽창률과 완전부화율은 EM grid, OPS 및 Cryo-loop 등과 같은 동결용기에 의해 큰 차이를 보이지 않았다. 또 Hoechst 염색에 의해 조사한 동결융해 후 체외에서 발달된 완전팽창 배반포의 총세포수에 있어서도, 대조군 (180.0 $\pm$ 5.4)과 동결군 (178.0 $\pm$ 7.5) 사이에 차이가 없었고, 동결융해 후 세포의 손상을 이중염색법으로 조사한 생존세포와 사멸세포의 비율도 대조군 (176 : 4)과 동결군 (172 : 6) 사이에 유의차가 인정되지 않았다. 이러한 결과로 보아 소 수정란은 EG 5.5 동결용액과 EM grid, OPS 또는 Cryo-loop과 같은 동결용기에 의해 성공적으로 동결보존할 수 있는 것으로 판단된다.

• Molecules and Cells
• /
• 제43권3호
• /
• pp.298-303
• /
• 2020

Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine high-resolution protein structures using the limited resource of cryo-EM instrument access.

• 한국수정란이식학회지
• /
• 제19권1호
• /
• pp.27-34
• /
• 2004

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.

• Molecules and Cells
• /
• 제46권9호
• /
• pp.538-544
• /
• 2023

The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.

• Clinical and Experimental Reproductive Medicine
• /
• 제30권3호
• /
• pp.241-248
• /
• 2003

Objective: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. Methods: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. Results: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. Conclusion: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.

• Clinical and Experimental Reproductive Medicine
• /
• 제25권1호
• /
• pp.71-76
• /
• 1998

본 연구는 소 미성숙난자를 electron microscope (EM) grid와 동해제인 EFS30을 이용하여 초급속 동결하였을 때 정상적인 배 발달의 유도가능성 여부를 조사하고자 실시하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 PBS에 첨가되어 제작된 EFS30을 사용하였다. 난자 생존의 평가기준으로는 성숙, 수정 및 배발달을 조사하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 소 미성숙란의 생존율은 43.2%을 나타내었다. 동결-융해군의 체외 성숙 (84.1%)과 정상 자웅전핵 형성율 (57.5%)은 대조군의 결과 (92.5, 65.0%)와 비교하여 볼 때 유의한 차이는 없었다. 또한, 동결군의 체외수정 이후의 $\geq2$-세포기 형성 (65.0%)과 배반포형성율 (30.8%)도 대조군(73.7, 35.7%)의 결과와 유의한 차이를 나타내지 않았다. 따라서 소 미성숙난자는 EM grid와 EFS30 동결액을 이용한 초급속 동결방법에 의해 정상적인 배발달이 유도될 수 있다는 것을 알 수 있었다.

• 마이크로전자및패키징학회지
• /
• 제26권3호
• /
• pp.81-88
• /
• 2019

본 연구에서는 그래핀 산화(graphene oxide, GO) 분말 첨가가 ball grid array(BGA) 패키지와 printed circuit board(PCB)간 Sn-3.0Ag-0.5Cu(SAC305) 무연솔더 접합부의 electromigration(EM) 수명에 미치는 영향에 대하여 보고 하였다. 솔더 접합 직후, Ni/Au표면처리된 패키지 접합계면에서는 $(Cu,Ni)_6Sn_5$가 생성되었으며 organic solderability preservative(OSP) 표면처리 된 PCB 접합계면에서는 $Cu_6Sn_5$ 금속간화합물(intermetallic compound, IMC)이 생성되었다. $130^{\circ}C$, $1.0{\times}10^3A/cm^2$ 전류밀도 하에서 EM 수명평가 결과, GO를 첨가하지 않은 솔더 접합부의 평균 파괴 시간은 189.9 hrs으로 도출되었고, GO를 첨가한 솔더 접합부의 평균 파괴 시간은 367.1 hrs으로 도출되었다. EM에 의한 손상은 패키지 접합계면에 비하여 pad 직경이 작은 PCB 접합계면에서 전자 유입에 의한 Cu의 소모로 인하여 발생하였다. 한편, 첨가된 GO는 하부계면의 $Cu_6Sn_5$ IMC와 솔더 사이에 분포하는 것을 확인하였다. 따라서, SAC305 무연솔더에 첨가된 GO가 전류 집중 영역에서 Cu의 빠른 확산을 억제하여 우수한 EM 신뢰성을 갖는 것으로 생각된다.

• Journal of electromagnetic engineering and science
• /
• 제2권1호
• /
• pp.16-21
• /
• 2002

On the contrary to the progress of the electronic industry and radio communication technologies, many social problems such as EMI, due to unnecessary electromagnetic(EM) wave are serious with the increased use of EM wave. It is required that the absorbing capability of an EM wave absorber is more than 20 dB, the bandwidth of which is required from 30 MHz to 18 GHz to satisfy the international standard about an anechoic chamber for EMI/EMS measurement$^{[1]}$TEX>. However, the absorbing frequency band of the conventional EM wave absorbers satisfying more than 20 dB is very narrow, for examples, from 30 MHz to 400 MHz in ferrite tile type and from 30 MHz to 870 MHz in ferrite grid type, respectively. In this paper, we proposed and designed a new tripe absorber with broadband characteristics covering the frequency band from 30 MHz to 10 GHz by use of the equivalent material constants method (EMCM)$^{[2]~[4]}$TEX>.

• 한국수정란이식학회지
• /
• 제16권3호
• /
• pp.233-237
• /
• 2001

To develop an effective vitrification method, we examined the use of a conventional straw as vessel fur vitrification of mouse oocytes, and to compare the post-thaw survival and chromosome configuration of these oocytes with those vitrified in grids. Intact cumulus-enclosed oocytes were vitrified with DPBS with 5.5 M ethylene glycol and 1.0 M sucrose, and loaded into straws and onto eletron microscopic copper grid fur storing in liquid nitrogen. Intact vitrified and thawed oocytes were karyotying for chromosome. The rates of post-thawed survival were 88.5% in vitrified oocytes with straws, and 83% in vitrified ooctyes with grids. Vitrified and thawed oocytes with straws and grids were increased chromosomal abnormality (31.4% and 30.9%) compared with fresh oocytes (17.8%). The conventional straws can be used as vessel for vitrification to prevent of inflection in liquid nitrogen.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.83-83
• /
• 2003

돼지 난자의 유리화 동결 처리 방법 중 난자를 담는 용기(loading vessel)의 재료로 최근에 알려진 것으로 open-pulled straws(OPS)[Vajta등, Mol Reprod Dev, 51:53-58, (1998)], electron microscope grids(EMG) [Martino등,Biol Reprod, 54:1059-1069, (1996)〕, nylon loop system(NLS) [Lane등, Fertil Steril,72: 1073-1078, (1999)] 등이 보고되고 있다. OPS는 1/4cc straws를 열을 가하여 길게 뽑아 내벽을 얇게 함으로써 filing된 난자나 수정란이 액체 질소와 접촉했을 때 유리화가 신속하게 되도록 하는 방법으로 돼지에서는 별로 보고된 것이 없다. EMG는 열전도가 예민한 전자현미경용 copper grid를 이용한 방법으로 최근 국내 기술진의 연구성적을 포함한 몇몇 학자들에 의하여 보고되었고 NLS는 0.5mm직경의 nylon loop를 이용하여 급속 동결한 성적이 보고되었으나, 돼지 난자에 응용 된 것은 없다. 따라서 이와 같은 동결 재료는 사람과 반추류, mouse외에 돼지 난자에 대하여는 전혀 시도되지 않았지만 유리화 동결기술에서 가장 중요한 실험으로 생각된다. 성공적인 유리화 동결을 위해서는 수정란이 냉각의 전도성이 빠르고, 작은 용액을 수정란과 같이 filling해야 하며 모든 동작이 신속 간편해야 하며 융해 방법도 초급속도의 융해가 요구되므로 이에 부합되어야 한다. 연구 목적은 돼지 난자를 유리화 동결/융해 시 동결 재료-straw/glass, copper grid, nylon 3가지에 대한 제작 방법, 난자 loading, 동결 처리, 보관 방법, 융해 방법 등을 난자의 회수, 수정 후 생존율을 비교 조사하여 가장 우수한 방법을 선택할 목적이었다. 수행 내용은 3가지의 재료의 sample을 제작하고 소독한 다음 준비된 돼지 COCs을 40시간동안 IVM한 후 난자를 5~l5개 정도로 선정 하여 준비된 VS 용액에 평형처리 하였다. 각 재료의 용기에 loading 한 후 동결/보관하였고, 융해는 역순으로 평형하여 maturation 배지에 3~4시간 배양한 다음 경검하고 IVF한 후 NCSU-23 배지에 담아 IVC 배양하면서 cell cleavage상태를 확인하였다.