• Applied Microscopy
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• 제42권2호
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• pp.111-114
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• 2012

The scanning electron microscopy is an ideal technique for examining plant surface at high resolution. Most hydrate samples, however, must be fix and dehydrate for observation in the scanning electron microscope. Because the microscopes operate under high vacuum, most specimens, especially biological samples, cannot withstand water removal by the vacuum system without morphological distortion. Cryo-techniques can observe in their original morphology and structure without various artifacts from conventional sample preparation. Rapid cooling is the method of choice for preparing plant samples for scanning electron microscopy in a defined physiological state. As one of cryo-technique, high-pressure freezing allows for fixation of native non-pretreated samples up to $200{\mu}M$ thick and 2 mm wide with minimal or no ice crystal damage for the freezing procedure. In this study, we could design to optimize structural preservation and imaging by comparing cryo-SEM and convention SEM preparation, and observe a fine, well preserved Arabidopsis stem's inner ultrastructure using HPF and cryo-SEM. These results would suggest a useful method of cryo-preparation and cryo-SEM for plant tissues, especially intratubule and vacuole rich structure.

• Applied Microscopy
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• 제39권1호
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• pp.65-72
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• 2009

Cryo-SEM은 수분을 함유하거나 액상 시료를 동결된 상태로 관찰하는 방법으로 rapid cooling, fracturing, sectioning, etching, coating과 같은 다소 복잡한 여러 과정의 전처리(Cryo-methods) 로 구성된다. 각 과정에서는 분석 목적이나 시료 특성(예: 시료크기, 물 함량 등)을 고려하여 최적의 조건을 설정하여야 한다. 본 연구에서는 cryo-SEM을 이용하여 남세균 (cyanobacteria, Synechocystis sp. PCC 6803)의 표면 및 내부 구조 관찰을 위한 etching time과 시료 처리에 관하여 살펴보았고 cryo-methods로 처리한 후 cryo-SEM으로 관찰한 이미지를 화학 고정한 일반 SEM(CSEM) 결과와 비교하였다. 관찰 결과, 화학 고정한 CSEM에서는 Synechocystis sp. PCC 6803 표면에 존재하는 pili가 잘 관찰되지 않았으며 파단된 내부 구조의 이미지를 얻을 수 없었으나 cryo-methods/cryo-SEM에서는 세포 표면의 pili 및 membrane의 형태를 관찰할 수 있었다. 또한 수화 상태에 따라 구조변형이 일어나는 biofilm의 구조도 관찰할 수 있었다. 이러한 결과로부터 cryo-methods/cryo-SEM은 수분을 함유하는 의생물 시료의 형태 구조를 분석하는 유용한 방법으로 사료된다.

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

• Archives of Plastic Surgery
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• 제40권4호
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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.

• Applied Microscopy
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• 제42권1호
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• pp.49-52
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• 2012

For TEM study of biological samples or polymers that are contained in organic structure, it is often required that the sample is prepared by using ultramicrotome and stained with proper agents to increase the contrast of organic structure. In this study, we investigated an efficient TEM sample preparation method for ductile heterogeneity material by using ultramicrotomy. Cryo-ultramicrotomy is a suitable method that is capable of rendering sample hardness for various ductile materials. However, it has several factors to consider, such as experimental cost, working time and finding the optimal staining conditions. To satisfy these considerations, we prepared TEM sample by using ultramicrotome without cryofunction, and secured the sample hardness by applying the staining process prior to ultrathin sectioning. The cross-linked polyethylene structure in the sample was stained with the 2% $RuO_4$ solution in a sealed test tube for 24 hours at $4^{\circ}C$. After the sample staining, ultrathin sections of sample were prepared using ultramicrotome. As a result, it was revealed that the difficulties associated with staining of ultrathin sections prepared by low-temperature conditions were improved. In addition, appropriate staining depth of sample could be selected for sectioning process. The quality of TEM sample obtained by using this method was better than that of cryo-ultramicroscopy. Finally, it is expected that our method could be effectively applied in TEM sample preparation for a variety of nano-bio convergence materials.

• Applied Microscopy
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• 제47권3호
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• pp.171-175
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• 2017

Negative staining has been traditionally used for exosome imaging; however, the technique is limited to surface topology only and can cause staining artifacts. Therefore, to analyze the internal structure of exosomes, we employed a method of block preparation, thin sectioning, and electron tomography. In addition, an automatic serial sectioning technique with 15-nm thickness through focused ion beam was employed to observe the three-dimensional structure of exosomes of various sizes. Cryo-transmission electron microscopy revealed the near-to-native structure of exosomes.

• Molecules and Cells
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• 제46권9호
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• pp.538-544
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• 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
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• 제45권4호
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• pp.177-182
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• 2018

Objective: To investigate the effects of in vitro myo-inositol (Myo-Ins) supplementation of cryopreserved human semen on the cryo-survival rate (CSR). Methods: Semen samples were obtained from 41 infertile men. Following routine semen analysis, each sample was divided into two equal aliquots (0.5 mL each). One aliquot was treated with 1 mg of Myo-Ins dissolved in $10{\mu}L$ of sperm preparation medium. The second aliquot was treated with $10{\mu}L$ of the same medium (control). Both aliquots were incubated for 20 minutes prior to freezing to slow the freezing process. The frozen samples were examined for post-thaw percentages of total motility (TM), progressive motility (PM), and the CSR, defined as the percentage of post-thaw TM divided by the percentage of pre-freeze TM and multiplied in 100. The results were expressed as median and interquartile range (25th and 75th percentiles). Results: The pre-freeze TM (50% [30%-50%]) and PM (35% [20%-35%]) were significantly higher than the post-thaw TM and PM in the MyoIns group (15% [10%-35%] and 10% [5%-20%]; p<0.001 and p<0.001, respectively) and the control group (10% [6%-30%] and 5% [3%-15%]; p<0.001 and p<0.001, respectively). The CSR of the 41 semen aliquots supplemented with Myo-Ins (40% [25%-70%]) was significantly higher than that of the control samples (30% [13%-58%], p=0.041). The CSR of the 26 abnormal semen samples that were supplemented with Myo-Ins (38% [20%-50%]) was significantly higher than that of the control samples (23% [12%-30%], p=0.031). Conclusion: In vitro Myo-Ins supplementation of ejaculated human sperm from infertile men resulted in a significant increase in the CSR in samples with abnormal pre-freeze sperm parameters.

• 한국수산과학회지
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• 제29권5호
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• pp.578-585
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• 1996

정어리, 명태 및 오징어육을 $-20^{\circ}C,\;-40^{\circ}C$및 $-80^{\circ}C$에서 동결하고 hammer mill에 의한 분쇄적성과 분쇄물의 mass colloider에 의한 마쇄 특성을 조사하였다. 정어리, 명태 및 오징어육은 $-40^{\circ}C$ 및 $-80^{\circ}C$로 동결할 경우 hammer mill에 의해 분쇄가 가능하였고 분쇄물은 mass colloider에 의해 마쇄가 가능한 것으로 나타났다. 어육별 동결분쇄물의 입도분포는 오징어, 정어리, 명태순으로 큰 입자의 비율이 높은 것으로 나타났다. 한편 동결분쇄물의 입자분포는 동결온도에 따라 차이를 보여 $-40^{\circ}C$ 보다는 $-80^{\circ}C$ 동결군의 입도가 작게 나타나는 경향을 보였다. 한편 동결온도가 다른 어육의 동결분쇄입자들에 대하여 image analysis를 행한 결과, $-40^{\circ}C$ 처리구는 짓이겨진 형태를 보인 반면 $-80^{\circ}C$ 처리구는 예리하게 끊어진 형태를 보였다. 또한 어종간의 조직특성이 관찰되어 어종 및 동결온도에 의해 동결분쇄물의 입도 분포 및 물성이 다를 수 있음이 시사되었다. 동결마쇄물의 수율은 정어리 $62.5\%$, 명태 $56.9\%$, 오징어 $52.5\%$로 나타났으며 오징어를 제외한 명태 및 정어리육의 마쇄물은 특히 뼈에서 유래되는 Ca의 함량비가 $6\~8$배 높은 특성을 보이고 있었다. 또한 동결마쇄물의 품질지표의 하나인 유화능을 측정한 결과, 대조군에 비해 다소 낮게 나타났으나 어육의 가공에는 충분히 적용가능할 것으로 평가되어 hammer mill과 mass colloider를 이용한 어육의 동결마쇄기법은 향후 관련분야에 이용성이 클 것으로 생각된다.

• 대한수의학회지
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• 제56권2호
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• pp.85-89
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• 2016

We tested a set of conditions for obtaining optimal tissue quality in preparation for histology in samples of mouse brain. C57BL/6J mice were sacrificed and perfused with 4% paraformaldehyde, after which the brains were removed and dehydrated in 30% sucrose solution. The brains were then divided into four groups according to freezing temperature and usage of optimal cutting temperature (OCT) compound. Next, we stained the sectioned brain tissues with Harris hematoxylin and eosin Y and immunohistochemistry was performed for doublecortin. The best quality tissue was obtained at $-25^{\circ}C$ and by not embedding with the OCT compound. When frozen at $-25^{\circ}C$, the embedded tissue was significantly damaged by crystals, while at $-80^{\circ}C$ there were no meaningful differences between qualities of embedded- and non-embedded tissues. Overall, we identified a set of conditions to obtain quality frozen brain sections. Our developed protocol will help resolve matters associated with damage caused to sectioned brain tissue by crystal formation during freezing.