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

点击翻译按钮获取中文摘要

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.