Acquisition of Cryo-Preparative Instrumentation for an Electron Microscopy Core

购买用于电子显微镜核心的冷冻制备仪器

• 批准号: 0215662
• 负责人: Thomas Phillips
• 金额: $ 14.44万
• 依托单位: University of Missouri-Columbia
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0215662&HistoricalAwards=false
• 关键词: Acquisition; Cryo; Preparative; Instrumentation; Electron

Phillips- 0215662A grant has been awarded to Dr. Thomas E. Phillips to fund the purchase of three cryo-preparative instruments: (i) a high-pressure freezing machine, (ii) an automated freeze-substitution and low temperature embedding processor, and (iii) a cryo-ultramicrotome. Conventional chemical fixation using aqueous fixatives cause unnatural changes in the fine structure of cells. Conventional fixation and embedding techniques at room temperature are also well known to interfere with immunocytochemical localization of target antigens. Cryo-fixation at ambient pressure results in the formation of ice-crystals that destroys the fine structure in all but the most superficial 10-20 mm of tissue. Freezing at very high pressure increases the depth of tissue without ice crystal damage to over 200 mm. The combination of cryo-fixation and freeze-substitution results in not only a truer preservation of tissue fine structure but also a greatly enhanced immunoreactivity. For samples which can not be fixed using high-pressure fixation, such as those collected in the field, conventional fixation followed by cryo-ultramicrotomy has been demonstrated to show much higher immunoreactivity than specimens dehydrated and embedded in plastic resins. To maximize the impact of this technology, these instruments will be housed in a multi-user electron microscopy facility for use by all researchers on our campus. Furthermore, investigators at other regional institutions will be able to use these instruments to prepare tissues that they can then examine on their home campuses. Examples of the type of research to be performed with this equipment include the Phillips laboratory's plan to freeze plant seed tissues to examine the pathway that both endogenous and exogenous transgenic proteins travel through plant cells. The Baskin laboratory studies how a plant organ attains a specific and heritable shape. High pressure freezing will allow this lab to define the role that the filaments of the cell's cytoskeleton play in determining the shape of the root. The sequencing of the complete or partial genomes of a growing number of bacteria, plant and animal species has created a wealth of information concerning what proteins are made by these different organisms. The next big breakthrough will be to determine the function of each of the proteins. The first steps in this process are determining in which cells the proteins are expressed and where exactly within the cell the proteins are located at different developmental or physiological stages. Electron microscopic immunocytochemistry offers a way to visualize the precise location of specific proteins and non-protein antigens within cells. These cryo-preparative instruments will not only increase the chances that our antibodies will recognize their target proteins but also ensure that the location that we find those proteins in accurately reflects their distribution in living cells.

Phillips-0215662A赠款已授予Thomas E. Phillips博士，以资助购买三种冷冻仪器的购买：（i）高压冰点机，（ii）自动化的冻结固体和低温嵌入处理器，以及（iii）Cryo-ultramicicirotome。 使用水性固定剂的常规化学固定会导致细胞的细胞结构不自然。 在室温下的常规固定和嵌入技术也众所周知，会干扰靶抗原的免疫细胞化学定位。 在环境压力下的冷冻固定会导致形成冰结晶，从而破坏了除最浅10-20毫米的组织以外的所有细胞结构。 在非常高压下的冰点会增加组织的深度，而没有冰晶体损伤至200毫米以上。 冷冻固定和冻结固定的结合不仅可以使组织细胞结构的真实保存，而且还可以极大地增强免疫反应性。 对于无法使用高压固定的样品（例如在田间收集的样品）中，已证明传统固定后，然后进行冷冻 - 粉状切开术的样品显示出比脱水并嵌入塑料树脂中的标本更高的免疫反应性。 为了最大程度地利用这项技术的影响，这些仪器将安置在多用户电子显微镜设施中，以供我们校园中的所有研究人员使用。 此外，其他区域机构的调查人员将能够使用这些工具来准备可以在家庭校园里检查的组织。 使用该设备进行的研究类型的例子包括菲利普斯实验室冻结植物组织组织的计划，以检查内源性和外源性转基因蛋白穿过植物细胞的途径。 巴斯金实验室研究植物器官如何达到特定而可遗传的形状。 高压冻结将使该实验室能够定义细胞细胞骨架的细丝在确定根部形状中的作用。 越来越多的细菌，动植物物种的完整或部分基因组的测序创造了有关这些不同生物所产生的蛋白质的大量信息。 下一个重大突破将是确定每种蛋白质的功能。 该过程中的第一步是确定蛋白质在哪些细胞中的表达以及蛋白质在细胞中的确切位置位于不同的发育或生理阶段。 电子显微镜免疫细胞化学提供了一种可视化细胞中特定蛋白质和非蛋白质抗原的精确位置的方法。这些排斥仪器不仅会增加我们抗体识别其靶蛋白的机会，而且还可以确保我们发现这些蛋白质的位置准确地反映了它们在活细胞中的分布。