TOMOGRAPHY OF FROZEN-HYDRATED TISSUE SECTIONS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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，从而提供了一个视图的标本？近本土？状态生物标本中的水必须被冻住吗？玻璃的？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。冷冻组织必须保持在低于去玻璃化温度（~-140 ℃）。C）整个超微切片术和显微镜检查。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。冷冻水化切片的断层扫描图显示出极好的结构保存，也表现出良好的切片质量，几乎没有表面伪影（裂缝）。这种相关性促使我们将早期断层扫描的电子衍射图案与新断层扫描的电子衍射图案进行比较，以测试以下假设：提高切割质量是由于高压冷冻后微晶冰含量较低。然而，在这两种情况下都没有观察到结晶冰的迹象。 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。我们发现切片与Quantifoil网格以及折叠网格连接，尽管在切片切割后不久必须检查网格，因为切片在长期储存中很容易丢失。 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。使用切片机？的玻璃切片压制工具，而不是抛光的金属棒，需要安全地压平Quantifoil网格上的切片。我们还发现，使用钼网格，减少了碳膜的扭曲，并可能有助于截面连接。 与RPI的Toh-Ming Lu博士合作，我们已经开始研究TEM网格的功能化涂层，这可能有助于冷冻水合切片更牢固地附着在网格上。在与RPI的Jay McMahon合作中，我们计划设计和制造特殊的两部分网格，这些网格可以牢固地夹紧，并有望使截面变平。

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 that showed excellent structural preservation and also exhibited good sectioning quality, with few surface artifacts (crevasses). This correlation prompted us to compare electron diffraction patterns of earlier tomograms with those the new ones, to test the hypotheses that the improved cutting quality was due to a lower content of microcrystalline ice after high-pressure freezing. However, signs of crystalline ice were not observed in either case. 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 were attached to Quantifoil grids as well as they were to folding grids, although the grids had to be examined shortly after the sections were cut since sections were easily lost in long-term storage. The thinner Quantifoil grids are advantageous for tomography because they allow more open area at high tilt. Use of the microtome?s glass section press tool, and not the polished metal rod, was required to safely flatten the sections on the Quantifoil grids. We also found that the use of molybdenum grids, reduced wrinkling of the carbon film and may aid in section attachment. In collaboration with Dr. Toh-Ming Lu of RPI, we have started investigating functionalized coatings for TEM grid that may help frozen-hydrated sections to attach more firmly to the grid. In collaboration with Jay McMahon of RPI, we plan to design and fabricate special two-part grids that firmly clamp, and hopefully flatten, the sections.