High Pressure Freezer for Improved Ultrastructure

用于改善超微结构的高压冷冻机

• 批准号: 6580180
• 负责人: DAVID H. HALL
• 金额: $ 19.39万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6580180
• 关键词: biomedical equipment purchase; cryoscience; freezing; tissue /cell preparation; transmission electron microscopy

DESCRIPTION (provided by applicant): The users assembled in this application have a common interest in preparing small samples for transmission electron microscopy. High Pressure Freezing (HPF) allows the fastest high quality fixation of tissue samples (200 microns thickness x 2mm diameter) for high resolution electron microscopy. Preliminary studies by the investigators have shown that we can produce "life-like" views in thin section of biological events such as endocytosis and glycocalyx changes which were rarely if ever seen previously by conventional chemical immersion fixation. Similar improvements can also be carried to high resolution immunocytochemistry, where enhanced tissue preservation is highly desirable. Many of these experiments will utilize immunogold techniques following HPF, freeze substitution and embedding in low temperature resins to localize proteins or mRNA in various tissues and cell types, all of which will benefit from improved spatial resolution. The grant is based upon funded projects in which this technique offers substantial improvements over current methods for animal cells, whole tissues, yeast and bacteria. Dr. Hall will be applying this technique to study mutations of the model organism Caenorhabditis elegans affecting tissue differentiation, including cytoskeleton, organelle integrity, and synaptic structure. The Major Users will utilize the machine to characterize the mitotic spindle in Drosophila embryos (Dr. Sharp), the structure of caveoli in yeast and in knockout mice (Dr. Lisanti), the distribution of mRNAs in yeast (Dr. Singer), and podocytes in the kidney glomerulus of knockout mice (Dr. Mundel). The machine can also prepare frozen samples for cryosectioning prior to cryeEM of the frozen sample. With this technique, Dr. Singer can conduct 3D tomographic reconstruction of the yeast Iocosome and Dr. Mundel will reconstruct the podocyte slit diaphragm.

描述（由申请人提供）： 在此应用程序中聚集的用户在制备透射电子显微镜的小样品方面有共同的兴趣。高压冷冻（HPF）允许最快的高质量固定组织样品（200微米厚x 2mm直径），用于高分辨率电子显微镜。研究人员的初步研究表明，我们可以在薄切片中产生“逼真”的生物事件，如内吞作用和糖萼变化，这是以前很少看到的常规化学浸泡固定。类似的改进也可以用于高分辨率免疫细胞化学，其中增强的组织保存是非常期望的。这些实验中的许多将利用免疫金技术在HPF、冷冻取代和低温树脂包埋之后定位各种组织和细胞类型中的蛋白质或mRNA，所有这些都将受益于提高的空间分辨率。该补助金是基于资助的项目，其中该技术提供了对动物细胞，整个组织，酵母和细菌的现有方法的实质性改进。霍尔博士将应用这种技术来研究影响组织分化的模式生物秀丽隐杆线虫的突变，包括细胞骨架，细胞器完整性和突触结构。主要用户将利用该机器表征果蝇胚胎中的有丝分裂纺锤体（Sharp博士）、酵母和基因敲除小鼠中的小窝结构（Lisanti博士）、酵母中mRNA的分布（Singer博士）以及基因敲除小鼠肾小球中的足细胞（Mundel博士）。该机器还可以在冷冻样品的cryeEM之前制备用于冷冻切片的冷冻样品。通过这项技术，Singer博士可以对酵母Iocosome进行3D断层扫描重建，Mundel博士将重建足细胞狭缝隔膜。