DEVELOPMENT OF CORRELATIVE LIGHT, EM APPROACH IN ISOLATED C ELEGANS EMBRYO

相关光、电磁方法在分离的秀丽隐杆线虫胚胎中的发育

• 批准号: 7355006
• 负责人: THOMAS MULLER-REICHERT
• 金额: $ 0.47万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355006
• 关键词: capillary; centrosome; lighting

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. Over the past three years, we have used whole worms prepared by high pressure freezing to image centrosomes in early embryos. This technique gave excellent ultrastructural preservation, but did suffer from a lack of time-resolution because the embryos showed variation in the mitotic stages. To overcome these problems, we have developed a correlative light and electron microscope approach that combines the benefit of time-resolution with the advantage of high-pressure freezing. Our approach is to collect single embryos into thin cellulose capillary tubes (Hohenberg et al., 1994) where they can be imaged alive under the light or fluorescence microscope. Embryos containing GFP:tubulin and GFP:hisotone constructs are imaged and filmed in order to monitor cells in different stages of mitosis. The staged embryos in the capillary tubes are then high-pressure frozen, freeze-substituted and embedded for electron tomography.This method gives specimens with excellent ultrastructural preservation of the mitotic centrosome. We have recently completed 9 tomograms from isolated wild-type embryos. This method will be used in future RNAi studies where imaging 1 or 2 cell embryos is essential.

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。在过去的三年里，我们使用高压冷冻制备的整个蠕虫来成像早期胚胎中的中心体。这种技术提供了极好的超微结构保存，但确实缺乏时间分辨率，因为胚胎在有丝分裂阶段显示出变化。为了克服这些问题，我们已经开发了一种相关的光和电子显微镜的方法，结合了时间分辨率的好处与高压冷冻的优势。我们的方法是将单个胚胎收集到薄的纤维素毛细管中（Hohenberg等人，1994年），其中它们可以在光或荧光显微镜下活体成像。对含有GFP：微管蛋白和GFP：hisotone构建体的胚胎进行成像和拍摄，以监测有丝分裂不同阶段的细胞。毛细管中的分阶段胚胎被高压冷冻，冷冻置换和包埋用于电子断层扫描，这种方法得到的标本具有良好的有丝分裂中心体的超微结构保存。我们最近完成了9个分离的野生型胚胎的断层扫描。这种方法将用于未来的RNAi研究，其中成像1或2细胞胚胎是必不可少的。