TOMOGRAPHY OF FROZEN-HYDRATED TISSUE SECTIONS

冷冻水合组织切片的断层扫描

• 批准号: 7598347
• 负责人: CHYONGERE HSIEH
• 金额: $ 3.44万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598347
• 关键词: 3-Dimensional; Area; Biological; Biological Preservation; Blood flow; Carbon; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Crystal Formation; Dehydration; Depth; Diamond; Electrons; Film; Freezing; Funding; Glass; Grant; Ice; Institution; Laboratories; Literature; Liver; Methods; Microscopic; Microscopy; Molybdenum; Morphologic artifacts; Plastics; Puncture biopsy; Rattus; Recommendation; Relative (related person); Research; Research Personnel; Resources; Source; Specimen; Staining method; Stains; Surface; Techniques; Temperature; Testing; Tissues; Tomogram; Ultramicrotomy; United States National Institutes of Health; Water; Work; abstracting; base; experience; improved; nanoscale; pressure; sample fixation; tomography; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Electron microscopic study of frozen-hydrated biological material avoids the necessity for chemical fixation, dehydration, and staining, and thus provides a view of the specimen in a near-native state. The water in biological specimens must be frozen in vitreous or amorphous form in order to avoid nanometer-scale damage to the specimen due to ice crystal formation. For tissue, the preferred method is high-pressure freezing, due to the depth of good freezing that can be obtained. The frozen tissue must be maintained below the de-vitrification temperature (~-140¿C) throughout ultramicrotomy and microscopy. Steady progress has been made over the past two decades in cutting frozen-hydrated sections. Although it remains a challenging task, improved cryo-ultramicrotomes and diamond knives, together with the collective experience of the few laboratories engaged in this work, provided a good starting point for investigators wishing to make use of frozen-hydrated sections. In April, 2002, we were the first laboratory to obtain electron tomograms of frozen-hydrated sections (from high-pressure frozen rat liver tissue). The most important finding was that the interior of the section was free of surface artifacts, thus good 3-D information could be obtained. " Hsieh, C.-E., Marko, M., Frank, J., and Mannella, C.A. 2002. Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol. 138:63-73. We did subsequent work in three areas: improvements in high-pressure freezing, comparison of frozen-hydrated and freeze-substituted material, and improvements in section attachment to grids. Rat liver tissue was frozen using needle biopsy kits, with which it was possible to freeze tissue within 40 sec of blood flow cessation. Some tissue was freeze-substituted and embedded in plastic. The main differences between the two techniques related to the relative contrast of cellular components. Tomograms of frozen-hydrated sections showed excellent structural preservation, which correlated with good sectioning quality, yielding sections with few surface artifacts (crevasses). We investigated means to reduce the irreversible compression that occurs in the sectioning direction. Based on recommendations in the literature, we started tested both an oscillating 35¿ cryo diamond knife and a 25¿ diamond knife, but no improvement in compression was seen in initial tests. One of the major problems with tomography of frozen-hydrated sections is poor attachment of the sections to the gird. This is due in large part to lack of section flatness, which we documented by low-magnification stereo pairs. We found that sections could be attached to Quantifoil grids by use of a glass press tool, and the thinner Quantifoil grids are advantageous for tomography because they allow more open area at high tilt. We also found that the use of molybdenum grids reduced wrinkling of the carbon film, and may aid in section attachment. We identified strategies for identifying suitable grid areas for tomography, thus increasing the yield of successful tomograms.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要： 冷冻水合生物材料的电子显微镜研究避免了化学固定，脱水和染色的必要性，因此提供了一个样品的视图。 近天然 状态 生物标本中的水必须被冻住 玻璃体 或无定形形式，以避免由于冰晶形成而对样品造成纳米级的损害。 对于组织，优选的方法是高压冷冻，因为可以获得良好的冷冻深度。 在整个超微切片术和显微镜检查过程中，冷冻组织必须保持在去玻璃化温度（~-140 ℃）以下。 过去二十年来，在切割冷冻水化切片方面取得了稳步进展。 虽然这仍然是一项具有挑战性的任务，但改进的冷冻超微切片机和金刚石刀，以及从事这项工作的少数实验室的集体经验，为希望利用冷冻水化切片的研究人员提供了一个良好的起点。 2002年4月，我们是第一个获得冷冻水化切片（来自高压冷冻大鼠肝组织）电子断层图像的实验室。 最重要的发现是，该部分的内部是免费的表面伪影，从而可以获得良好的三维信息。 " 谢角，越-地E、Marko，M.，弗兰克，J，和Mannella，C.A. 2002. 冷冻水合组织切片的电子断层扫描分析。J.Struct.Biol.138：63-73. 我们在三个方面做了后续工作：高压冷冻的改进，冷冻水化和冷冻替代材料的比较，以及网格截面附着的改进。 使用穿刺活检套件冷冻大鼠肝组织，使用该套件可以在血流停止的40秒内冷冻组织。 一些组织被冷冻替代并嵌入塑料中。 这两种技术之间的主要差异与细胞成分的相对对比度有关。 冷冻水合切片的断层扫描显示出出色的结构保存，这与良好的切片质量相关，产生的切片表面伪影（裂缝）很少。 我们调查的手段，以减少不可逆的压缩，发生在切片方向。 根据文献中的建议，我们开始测试振荡35 <$cryo金刚石刀和25 <$金刚石刀，但在初始测试中没有看到压缩的改善。 冻结水化截面的层析成像的主要问题之一是截面与梁的附着不良。 这在很大程度上是由于缺乏截面平坦度，我们记录了低放大率立体对。 我们发现，可以通过使用玻璃压制工具将切片连接到Quantifoil网格上，并且更薄的Quantifoil网格有利于断层扫描，因为它们在高倾斜时允许更多的开放区域。 我们还发现，钼网格的使用减少了碳膜的扭曲，并可能有助于截面连接。 我们确定的战略，确定合适的网格区域断层扫描，从而提高成功的断层图像的产量。