Studying kinetochore-fiber ultrastructure using correlative light-electron microscopy

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Abstract

Electron microscopy (EM) has dominated high-resolution cellular imaging for over 50 years thanks to its ability to resolve on a nanometer-scale intracellular structures such as the microtubules of the mitotic spindle. It is advantageous to view the cell of interest prior to processing the sample for EM. Correlative light electron microscopy (CLEM) is a technique that allows one to visualize cells of interest by light microscopy (LM) before being transferred to EM for ultra-structural examination.

Here we describe how CLEM can be applied as an effective tool to study the spindle apparatus of mitotic cells. This approach allows transfected cells of interest, in desirable stages of mitosis, to be followed from LM through to EM. CLEM has often been considered as a technically challenging and laborious technique. In this chapter we provide step-by-step pictorial guides that allow successful CLEM to be achieved. In addition we explain how it is possible to vary the sectioning plane, allowing spindles and microtubules to be analyzed from different angles, and the outputs that can be obtained from these methods when applied to the study of kinetochore fiber (K-fiber) ultrastructure.

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