For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/). Key features • High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM). • Serial-section electron tomography of selected cells. • Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization.
Keywords: 3D reconstruction; Automated spatial graph analysis; Automatic segmentation of microtubules; CLEM; Correlative light and electron microscopy; Electron microscopy; Electron tomography; HeLa cells; Human cells; Microtubules; Mitotic spindle; Serial sectioning; Tissue culture cells.
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