TOMOGRAPHY OF FROZEN-HYDRATED TISSUE SECTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 对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，从而提供了标本处于“近乎原生”状态的视角。为了避免冰晶形成对标本造成纳米级的破坏，生物标本中的水必须冻结为“玻璃体”或无定形。对于组织，首选的方法是高压冷冻，因为可以获得良好的冷冻深度。在整个超微切割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。 在过去的二十年里，在切割冷冻水化部分方面取得了稳步进展。尽管这仍然是一项具有挑战性的任务，但改进的冷冻超微切割机和钻石刀具，再加上从事这项工作的少数几个实验室的集体经验，为希望利用冷冻水合切片的调查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)的电子断层图像的实验室。最重要的发现是，剖面内部没有表面伪影，因此可以获得良好的三维信息。 谢志荣，Marko，M.，Frank，J.及Mannella，C.A.2002。冷冻水合组织切片的电子断层扫描分析。J·斯特鲁特。比奥尔。138：63-73。 我们在三个方面做了后续的工作：高压冷冻的改进，冷冻水化材料和冷冻替代材料的比较，以及网格截面连接的改进。 大鼠的肝组织用针刺活检试剂盒冷冻，用这种试剂盒可以在血流停止后40秒内冷冻组织。一些组织被冷冻替代并嵌入塑料中。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。 冷冻水化切片的断层图像显示良好的结构保存，这与良好的切片质量相关，产生的切片表面伪影(裂缝)很少。我们研究了减少在截面方向上发生的不可逆压缩的方法。根据文献中的建议，我们开始测试摆动35°低温钻石刀和25°钻石刀，但在最初的测试中没有看到压缩方面的改善。 冷冻水合切片层析成像的主要问题之一是切片与网格的附着性差。这在很大程度上是由于缺乏截面平坦度，这是我们用低倍立体声对记录的。我们发现，可以使用玻璃压合工具将部分连接到量子箔网格上，较薄的量子箔网格对层析成像是有利的，因为它们允许在高倾斜时有更多的开放区域。我们还发现，钼网的使用减少了碳膜的皱折，并可能有助于截面附着。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： 谢春娥、Marko，M.，Leith，A.，Mannella，C.A.和Frank，J.(2006)对天然哺乳动物组织的高分辨率三维成像：冷冻水化大鼠肝脏切片的电子断层扫描。J·斯特鲁特。比奥尔。153(1)：1-13。 Marko，M.，Hsieh，C.E.，and Mannella，C.A.(2006)用于细胞和组织的电子断层扫描的冷冻水化切片。收信人：埃德。J·弗兰克，细胞和组织的电子断层摄影术，施普林格。 我们参加了2006年5月21-23日在德国慕尼黑附近的Schlos Hohenkammer举行的第三届玻璃体冷冻标本切片国际会议。在那里，我们了解了其他实验室在部分连接问题上的进展。一种方法是直接在切片上应用标记(以量子点的形式)。另一种方法是使用静电来帮助将部分连接到支撑膜上。我们正在研究这两种方法，认识到每种方法的局限性，特别是在厚的部分和有裂缝的部分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的陆东明博士的合作工作。我们已经对用于电子显微镜网格的功能化涂层进行了初步调查，以帮助实现截面连接，但结果并不确定，我们决定RPI工作应集中在TRD2上。 我们介绍了几个场馆冷冻水化切片的层析成像工作，主要是结合我们关于纤维球磨的工作(见下文)。其中一次演讲主要集中在低温超微切片上： 谢春娥，Mannella，C.，Ting，C.，Stokes，D.，Frank，J.，Marko，M.(2006)冷冻组织和细胞水合切片的电子断层扫描。程序第16届国际显微镜大会，H.Ichinose和T.Sasaki主编。2：691。 关于冷冻断层扫描，虽然不是冷冻水合部分，但出版了以下章节： Marko M.和Hsieh-E.(2006)细胞和细胞器的三维冷冻电子显微镜。收信人：埃德。J·郭，梅斯。摩尔。比奥尔。117.

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.