Acquisition of Cryo-Preparative Instrumentation for an Electron Microscopy Core

Phillips- 0215662A 拨款已授予 Thomas E. Phillips 博士，用于资助购买三台冷冻制备仪器：(i) 高压冷冻机，(ii) 自动冷冻替代和低温包埋处理器，以及 (iii) 冷冻超薄切片机。 使用水性固定剂的传统化学固定会导致细胞精细结构发生不自然的变化。 众所周知，室温下的常规固定和包埋技术也会干扰靶抗原的免疫细胞化学定位。 环境压力下的冷冻固定会导致冰晶的形成，从而破坏除最表层 10-20 毫米组织外的所有组织的精细结构。 高压冷冻可将组织深度增加至 200 毫米以上，而不会造成冰晶损伤。 冷冻固定和冷冻替代的结合不仅可以更真实地保存组织精细结构，而且可以大大增强免疫反应性。 对于无法使用高压固定来固定的样品，例如在现场收集的样品，已证明传统固定后进行冷冻超薄切片比脱水并包埋在塑料树脂中的样品显示出更高的免疫反应性。 为了最大限度地发挥这项技术的影响，这些仪器将安装在多用户电子显微镜设施中，供我们校园的所有研究人员使用。 此外，其他地区机构的研究人员将能够使用这些仪器来准备组织，然后在自己的校园中进行检查。 使用该设备进行的研究类型的例子包括菲利普斯实验室计划冷冻植物种子组织，以检查内源和外源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获得特定且可遗传的形状。 高压冷冻将使该实验室能够确定细胞骨架的细丝在确定根的形状方面所起的作用。 对越来越多的细菌、植物和动物物种的完整或部分基因组进行测序，产生了有关这些不同生物体产生哪些蛋白质的丰富信息。 下一个重大突破将是确定每种蛋白质的功能。 该过程的第一步是确定蛋白质在哪些细胞中表达，以及蛋白质在不同发育或生理阶段的确切位置。 电子显微镜免疫细胞化学提供了一种可视化细胞内特定蛋白质和非蛋白质抗原的精确位置的方法。这些冷冻制备仪器不仅会增加我们的抗体识别其目标蛋白质的机会，而且还能确保我们找到这些蛋白质的位置准确反映它们在活细胞中的分布。

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.