Visualization of centrosome biogenesis in Drosophila egg extract. (A) Drosophila egg extract is prepared by rupturing the membrane and aspirating the cytoplasm with a micropipette. The content is deposited as a droplet on functionalized glass surface. (B) Each explant is followed by 3D time-lapse imaging, documenting centriole formation over time. (C) Maximum intensity z projections from a fluorescence time-lapse of a droplet of cytosolic extract isolated from a Drosophila egg overexpressing Plk4. Centrioles are absent in the first time point and form de novo throughout the experiment detected as spots (Spd2, in green) associated with a MT array (magenta; arrowheads, numbers indicate the order of birth), reported by the MT associated protein Jupiter. Signals in the two channels are detected almost simultaneously, without observing any clear trend of one signal appearing before the other one. The larger green circles are yolk, and the high background is caused by other lipid granules that are highly autofluorescent in the green spectrum, and that cannot be avoided. The insets depict the first centrosomes formed de novo in this time-lapse. The numbers represent their order of appearance. Example of n = 68 explants. Time is reported as minutes:seconds.