TOMOGRAPHY OF FROZEN-HYDRATED TISSUE SECTIONS

冷冻水合组织切片的断层扫描

• 批准号: 7954571
• 负责人: CHYONGERE HSIEH
• 金额: $ 1.12万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954571
• 关键词: 3-Dimensional; Area; Biocompatible Materials; Biological; Biological Preservation; Blood flow; Book Chapters; Carbon; Cells; Charge; Chemicals; Collection; Computer Retrieval of Information on Scientific Projects Database; Congresses; Cryoelectron Microscopy; Crystal Formation; Dehydration; Diamond; Electrons; Electrostatics; Film; Freezing; Funding; Germany; Glass; Grant; Ice; Imagery; Institution; International; Investigation; Laboratories; Learning; Literature; Liver; Methods; Microscopic; Microscopy; Molybdenum; Morphologic artifacts; Needle biopsy procedure; Organelles; Plastics; Publications; Publishing; Quantum Dots; Rattus; Recommendation; Relative (related person); Reporting; Research; Research Personnel; Resolution; Resources; Series; Source; Specimen; Staining method; Stains; Surface; Techniques; Temperature; Testing; Thick; Three-Dimensional Imaging; Tissues; Tomogram; Ultramicrotomy; United States National Institutes of Health; Water; Work; abstracting; base; electron tomography; experience; improved; meetings; 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. We summarized the findings of the last reporting period in two publications: + Hsieh, C.-E., Marko, M., Leith, A., Mannella, C.A. and Frank, J. (2006) Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol. 153(1):1-13. + Marko, M., Hsieh, C.-E., and Mannella, C.A. (2006) Frozen-hydrated sections for electron tomography of cells and tissue. In: Ed. J. Frank, Electron tomography of cells and tissue, Springer. We attended the third international meeting on sectioning of vitreously-frozen specimens, held at Schloss Hohenkammer, near Munich, Germany on May 21-23, 2006. There, we learned of progress in other labs regarding the section attachment problem. One approach consisted of application of markers (in the form of quantum dots) directly on sections. Another approach used an electrostatic charge to help attachment of sections to a support film. We are investigating both approaches, realizing the limitations of each, especially in the case of thick sections and sections with crevasses that may deform during tilt-series collection. Because of this work, we are taking a hiatus from the collaborative work with Dr. Toh-Ming Lu of RPI. We had made preliminary investigations of functionalized coatings for TEM grids to aid in section attachment, but results were inconclusive and we decided that the RPI effort should be concentrated on TRD2. We presented the work on tomography of frozen-hydrated sections at several venues, mostly combined with our work on FIB-milling (see below). One talk concentrated mainly on cryo-ultramicrotome sections: + Hsieh, C.-E., Mannella, C., Ting, C., Stokes, D., Frank, J., Marko, M. (2006) Cryo electron tomography of frozen hydrated sections of tissue and cells. Proc. 16th International Congress on Microscopy H. Ichinose and T. Sasaki, eds. 2:691. Related to cryo-tomography, although not frozen-hydrated sections, the following book chapter was published: + Marko, M. and Hsieh, C.-E. (2006) 3-D cryo-electron microscopy of cells and organelles. in: Ed. J. Kuo, Meth. Mol. Biol. 117.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 抽象的： 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，从而提供了“接近天然”状态的样本视图。 生物样本中的水必须以“玻璃态”或无定形形式冻结，以避免由于冰晶形成而对样本造成纳米级的损坏。 对于组织，首选方法是高压冷冻，因为可以获得良好的冷冻深度。 在整个超薄切片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°C) 以下。 过去二十年来，冷冻水合切片的切割取得了稳步进展。 尽管这仍然是一项具有挑战性的任务，但改进的冷冻超薄切片机和金刚石刀，以及从事这项工作的少数实验室的集体经验，为希望利用冷冻水合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合切片（来自高压冷冻大鼠肝组织）的电子断层图的实验室。 最重要的发现是截面内部没有表面伪影，因此可以获得良好的 3D 信息。 + Hsieh, C.-E.、Marko, M.、Frank, J. 和 Mannella, C.A. 2002.冷冻水合组织切片的电子断层扫描分析。 J.结构。生物。 138：63-73。 我们在三个领域进行了后续工作：高压冷冻的改进、冷冻水合材料和冷冻替代材料的比较以及网格切片附着的改进。 使用针吸活检套件冷冻大鼠肝组织，可以在血流停止后 40 秒内冷冻组织。 一些组织被冷冻替代并嵌入塑料中。 两种技术之间的主要区别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良好的结构保存，这与良好的切片质量相关，产生的切片几乎没有表面伪影（裂缝）。 我们研究了减少切片方向上发生的不可逆压缩的方法。 根据文献中的建议，我们开始测试振荡 35° 冷冻金刚石刀和 25° 金刚石刀，但在初始测试中没有看到压缩方面的改善。 冷冻水合切片断层扫描的主要问题之一是切片与网格的附着不良。 这在很大程度上是由于缺乏截面平坦度，我们通过低放大倍数立体对记录了这一点。 我们发现可以使用玻璃压制工具将切片附着到 Quantifoil 网格上，并且较薄的 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，使用钼网格可以减少碳膜的褶皱，并可能有助于切片附着。 我们确定了确定适合断层扫描的网格区域的策略，从而提高了成功断层扫描的产量。 我们在两份出版物中总结了上一个报告期的调查结果： + Hsieh, C.-E.、Marko, M.、Leith, A.、Mannella, C.A. Frank, J. (2006) 对天然哺乳动物组织进行高分辨率三维成像：冷冻水合大鼠肝脏切片的电子断层扫描。 J.结构。生物。 153（1）：1-13。 + Marko, M.、Hsieh, C.-E. 和 Mannella, C.A. (2006) 用于细胞和组织电子断层扫描的冷冻水合切片。在：埃德。 J. Frank，细胞和组织电子断层扫描，施普林格。 我们参加了 2006 年 5 月 21 日至 23 日在德国慕尼黑附近的 Schloss Hohenkammer 举行的第三届玻璃冷冻标本切片国际会议。在那里，我们了解了其他实验室在切片附着问题方面的进展。 一种方法包括直接在切片上应用标记（以量子点的形式）。 另一种方法使用静电荷来帮助将切片附着到支撑膜上。 我们正在研究这两种方法，认识到每种方法的局限性，特别是在厚切片和带有裂缝的切片在倾斜系列收集过程中可能变形的情况下。 由于这项工作，我们暂停了与 RPI 的 Toh-Ming Lu 博士的合作。 我们对 TEM 网格的功能化涂层进行了初步研究，以帮助截面附着，但结果尚无定论，我们决定 RPI 工作应集中在 TRD2 上。 我们在多个地点展示了冷冻水合切片断层扫描的工作，大部分与我们在 FIB 铣削方面的工作相结合（见下文）。 一场演讲主要集中在冷冻超薄切片机切片上： + Hsieh, C.-E.、Mannella, C.、Ting, C.、Stokes, D.、Frank, J.、Marko, M. (2006) 组织和细胞冷冻水合切片的冷冻电子断层扫描。过程。第 16 届国际显微镜大会 H. Ichinose 和 T. Sasaki，编辑。 2:691。 与冷冻断层扫描相关，尽管不是冷冻水合切片，但出版了以下书籍章节： + Marko, M. 和 Hsieh, C.-E。 (2006) 细胞和细胞器的 3-D 冷冻电子显微镜。在：埃德。 J.郭，梅斯。摩尔。生物。 117.