Preparation of cytoskeletons of cells in culture for high resolution scanning and scanning transmission electron microscopy.

In this tutorial we describe our methods for preparing detergent-extracted cytoskeletons for observation by high resolution scanning (SEM) and scanning transmission (STEM) electron microscopy. We both discuss the theoretical background and provide practical procedures for each of the following steps: cell culture on Formvar-coated gold grids; prefixation with aldehydes or protein crosslinking reagents (homobifunctional N-hydroxy-succinimide esters); extraction with Triton X-100 or Brij 58 detergent in microtubule stabilizing buffer; postfixation in formaldehyde and glutaraldehyde; dehydration; critical point and freeze drying; sputter coating with 1-2 nm of platinum or tungsten; and examination by SEM and both normal and inverted contrast STEM. These methods produce cytoskeletal preparations in which filaments as fine as 7 nm are preserved and can be observed by scanning electron microscopy.