TOMOGRAPHY OF FROZEN-HYDRATED TISSUE SECTIONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。文摘：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，从而提供了一种近乎原生的标本的视角。州政府。生物标本中的水必须冻结在玻璃体中。或无定形，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，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，冰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结构保存良好，切片质量也很好，表面伪影(裂缝)很少。这种相关性促使我们将早期断层图像的电子衍射图与新断层图像的电子衍射图进行比较，以验证以下假设：切割质量的改善是由于高压冷冻后微晶冰的含量较低。然而，在这两个案例中都没有观察到结晶冰的迹象。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，截面连接到量子箔网格以及它们连接到折叠网格，尽管网格必须在截面被切割后不久进行检查，因为截面在长期存储中很容易丢失。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阔区域。要安全地压平量子箔网格上的截面，需要使用Microtome？S玻璃截面压制工具，而不是抛光的金属棒。我们还发现，使用钼网，减少了碳膜的皱折，并可能有助于断面附着。与RPI的陆拓明博士合作，我们已经开始研究用于透射电子显微镜网格的功能化涂层，这种涂层可能有助于冷冻水合部分更牢固地附着在网格上。与RPI的Jay McMahon合作，我们计划设计和制造特殊的两部分网格，牢牢地夹住并有望将部分压平。

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.