Time-resolved analysis of cytokinesis by correlative light and electron microscopy. (A) Timing of abscission events during the first embryonic division. The start of cleavage furrow ingression is defined as the 0 s time point. Single time points for each analyzed abscission event are indicated by symbols. A red line highlights the mean time point for each. The time point of abscission was obtained by tomographic analysis as shown in C. n = 11–15, depending on the step analyzed. (B) Determination of the time point of microtubule disassembly. Analysis of fluorescence intensity (mCherry::β-tubulin) in the region of the cleavage furrow (position 1) and in the cytoplasm (defined as background; position 2). The background fluorescence was subtracted from the fluorescence intensity at the cleavage furrow. The mean values of 10 single embryos were plotted over time. The time point of microtubule (MT) disassembly is indicated (red line). Error bars show SD. r.u., relative units. (C) 3D reconstruction of intercellular bridges at defined time points after initiation of furrow ingression as indicated in the right panels (top right corner, seconds). A light microscopy (LM) image (first column, GFP::PH in green and mCherry::β-tubulin in red), an electron microscopy image (second column, tomographic slice), the corresponding 3D model (third column), and a cartoon illustration (fourth column) are shown for each time point. Color coding is as follows: gold, plasma membrane of an open side of the intercellular bridge; red, microtubules; white, open microtubule ends; blue, closed microtubule ends; green, vesicles; dark gray background, cytoplasm at the open bridge; light gray background, cytoplasm at the disconnected bridge; nude, anterior cell membrane; and purple, posterior cell membrane. The midbody remnants (765 s and 900 s) are also shown in gold. (D) Additional tomographic z slices of an embryonic intercellular bridge (650 s; a thickening of the membrane is indicted by arrowheads). Bars, 500 nm.