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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在过去的三年里，我们用高压冷冻的整条蠕虫来成像早期胚胎中的中心体。该技术提供了极好的超微结构保存，但由于胚胎在有丝分裂阶段表现出变异，因此确实存在时间分辨率不足的问题。为了克服这些问题，我们开发了一种相关的光学和电子显微镜方法，结合了时间分辨率的优点和高压冷冻的优点。我们的方法是将单个胚胎收集到薄的纤维素毛细管中（Hohenberg et al., 1994），在那里它们可以在光或荧光显微镜下成像。含有GFP：微管蛋白和GFP：组蛋白结构的胚胎被成像和拍摄，以监测细胞在有丝分裂的不同阶段。在毛细管中发育的胚胎随后被高压冷冻，冷冻替代，并嵌入电子断层扫描。这种方法使有丝分裂中心体的标本具有良好的超微结构保存。我们最近完成了9个分离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个细胞胚胎是必不可少的。

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.