• Applied Microscopy
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• v.39 no.1
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• pp.65-72
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• 2009

Cryo-SEM which enables specimens to be observed in frozen form has been used to study liquid specimens in their native states. Cryo-methods, sample preparation for cryo-SEM, are quite complex and involve several discrete but vitally interconnected steps which are rapid cooling, fracturing, sectioning, etching and coating. It is important to select practical techniques and to optimize conditions of each steps considering analytic purpose and specimen characters, viz., sample dimension, water contents. In this study, etching methods and sample preparation before freezing had been studied for observation of cyanobacteria, Synechocystis sp. PCC 6803 using cryo-SEM and their cryo-SEM images were compared to Conventional SEM (CSEM) images treated by chemical fixation. We could observe the improved morphological images of the pili of the surface and membranes of Synechocystis sp. PCC 6803 and the three-dimensional architectures of their biofilm, which were difficult to observe using chemical fixation and CSEM. These results suggest that cryo-methods/cryo-SEM are useful techniques for morphological study of biological specimen.

• Applied Microscopy
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• v.48 no.1
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• pp.6-10
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• 2018

Cryo-electron microscopy (cryo-EM) allows the analysis of the near-native structures of samples such as proteins, viruses, and sub-cellular organelles at the sub-nano scale. With the recent development of analytical methods, this technique has achieved remarkable results. The importance of cryo-EM gained wide recognition due to last year's award of the Nobel Prize in Chemistry. To help promote the knowledge of this technique, this paper introduces the basic workflows of cryo-EM and domestic cryo-EM service institutes.

• Applied Microscopy
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• v.47 no.4
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• pp.223-225
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• 2017

Cryo-electron microscopy (cryo-EM) is arguably the most powerful tool used in structural biology. It is an important analytical technique that is used for gaining insight into the functional and molecular mechanisms of biomolecules involved in several physiological processes. Cryo-EM can be separated into the following three groups according to the analytical purposes and the features of the biological samples: cryo-electron tomography (cryo-ET), cryo-single-particle reconstruction, and cryo-electron crystallography. Cryo-tomography is a unique EM technique that is used to study intact biomolecular complexes within their original environments; it can provide mechanistic insights that are challenging for other EM-methods. However, the resolution of reconstructed three-dimensional (3D) models generated by cryo-ET is relatively low, while single-particle reconstruction can reproduce biomolecular structures having near-atomic resolution without the need for crystallization unless the samples are large (>200 kDa) and highly symmetrical. Cryo-electron crystallography is subdivided into the following two categories according to the types of samples: one category that deals with two-dimensional (2D) crystalline arrays and the other category that uses 3D crystals. These two categories of electron-crystallographic techniques use different diffraction data obtained from still diffraction and continuous-rotation diffraction. In this paper, we review crystal-based cryo-EM techniques and focus on the recently developed 3D electron-crystallographic technique called microcrystal-electron diffraction.

• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.13.1-13.7
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• 2016

Background: In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated. Methods: EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos. Results: It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant. Conclusions: In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen.

• Archives of Plastic Surgery
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• v.40 no.4
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• pp.374-379
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• 2013

Background To date, various types of acellular dermal matrix (ADM) have been developed for clinical use. AlloDerm is the most familiar type of ADM to most surgeons in breast reconstruction. It is prepared by freeze-drying. CG CryoDerm is the first form of ADM that requires no drying process. Therefore, theoretically, it has a higher degree of preservation of the dermal structures than AlloDerm. We conducted this study to compare the clinical course and postoperative outcomes of patients who underwent direct-to-implant breast reconstructions using AlloDerm and those who did using CG CryoDerm. Methods We performed a retrospective analysis of the medical records in a consecutive series of 50 patients who underwent direct-to-implant breast reconstruction using AlloDerm (n=31) or CryoDerm (n=19). We then compared the clinical course and postoperative outcomes of the two groups based on the overall incidence of complications and the duration of drainage. Results The mean follow-up period was 16 months. There were no significant differences in the overall incidence of complications (seroma, infection, skin flap necrosis, capsular contracture, and implant loss) between the two groups. Nor was there any significant difference in the duration of drainage. Conclusions CG CryoDerm has the merits of short preparation time and easy handling during surgery. Our results indicate that CG CryoDerm might be an alternative allograft material to AlloDerm in direct-to-implant breast reconstruction.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.63-69
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• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• Tuberculosis and Respiratory Diseases
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• v.66 no.2
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• pp.110-115
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• 2009

Background: A forceps-biopsy is performed to acquire tissue from patients with an endobronchial carcinoma using a flexible bronchoscope. Recently, a cryo-biopsy has also been used to acquire tissue samples. Cryo-biopsy is the diagnostic application of extreme cold for the local destruction of abnormal living tissue. This technique is safe, with no radiation danger, no risk of electrical accidents, and a little risk of bleeding. This study compared a forceps-biopsy with a cryo-biopsy using a flexible bronchoscope, and examined the chemosensitivity and level of VEGF (vascular endothelial growth factor) in the specimens obtained from the cryo-biopsy. Methods: We present a prospective study of 30 consecutive patients who underwent a forceps-biopsy between January 2007 and October 2007 with a mean age of 62.1 years and a male:female ratio of 5 : 1. A flexible bronchoscope was inserted to the area of the abnormal lesions, and a cryo-probe was then applied through the working channel of the flexible bronchoscope. A temperature of approximately -h80 was delivered to the tumor site for 8 seconds. The cryo-biopsy was performed after destroying the tumor mass. Results: The mean size of the tissue from the forceps-biopsy and cryo-biopsy were 2.0${\pm}$1.2 mm and 6.0${\pm}$3.0 mm. A chemosensitivity test was performed on 5 specimens obtained using cryo-biopsy and the level of VEGF was examined in 2 specimens obtained from a cryo-biopsy. There were no side effects in either group. Conclusion: Cryo-biopsy using a flexible bronchoscope is a safe and effective technique for acquiring tissue samples.

• Journal of Chest Surgery
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• v.28 no.8
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• pp.731-741
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• 1995

Cardiac valve allografts have been used as replacements for diseased valves and right ventricular outflow tract reconstruction, the long term follow-up of which has been reported satisfactory. For a good long-term result, it is essential that the allograft be viable at implantation. In this study, we aimed at preparing the cardiac valve allografts aseptically, preserving them at cold- and cryo-conditions, and testing the viability of the allografts after preservation by four methods. We tested the viability of the cardiac valve allografts preserved in cold refrigerated state[4$^{\circ}$C in nutrient media & in liquid nitrogen tank[cryopreservation under -149$^{\circ}$C for pre-planned time periods. The testing methods were 1 glucose utility test 2 tissue culture 3 thymidine uptake test and 4 histologic evidence by light microscopy. We observed no differences in the viability between cold- & cryo-groups and similar results among the methods for testing the viability. In conclusion, there was no difference in the viability between cold- and cryopreserved-allografts at least for 14 days of preservation. And glucose utility test and thymidine uptake test were satisfactory in the evaluation of the allograft viability, since they were easy and rapid with relatively quantitative results.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.