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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在过去的三年里，我们使用了高压冷冻制备的整虫来成像早期胚胎中的中心体。这项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势和高压冷冻的优势。我们的方法是将单个胚胎收集到细纤维毛细管中(Hohenberg等人，1994)，在那里它们可以在光学或荧光显微镜下被活生生地成像。含有GFP：微管蛋白和GFP：异构体的胚胎被成像和拍摄，以监测处于有丝分裂不同阶段的细胞。毛细管中的分期胚胎随后被高压冷冻、冷冻替代并包埋进行电子断层扫描。这种方法使标本具有良好的有丝分裂中心体的超微结构保存。我们最近已经完成了9个来自分离的野生型胚胎的断层扫描。这种方法将在未来的RNAi研究中使用，在那里成像1或2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.