IMAGING OF CYTOSKELETAL ELEMENTS BY LOW-TEMPERATURE HIGH-RESOLUTION SCANNING ELECTRON-MICROSCOPY

Actin filaments and microtubules, both in situ and in vitro, were imaged using high-resolution scanning electron microscopy (HRSEM) at low temperature, For visualization of cytoskeletal elements in situ, fibroblasts were first extracted and fixed; for cytoskeletal elements in vitro, purified proteins were polymerized and fixed. Both types of specimen were then subjected to plunge freezing, controlled freeze-drying, cryo-sputter coating with a thin chromium layer, cryo-transferring and cryo-observation in an FESEM. The three-dimensional architecture of the cytoskeleton was well preserved, permitting examination of the structural relationships among cytoskeletal elements. Actin filaments and microtubules were identified by their characteristic helical features. Two periodicities of actin filaments, the short pitch of the left-handed helix measured at 5.5 nm and the 37-nm-long pitch helix, were revealed. Individual protofilaments were seen in microtubules as well as the characteristic 4-nm repeat of tubulin subunits along the protofilament. Clathrin cages were also observed. This technique provides a powerful approach for direct imaging of macromolecular structures with high contrast and high signal-to-noise ratio at a resolution of 2-3 nm.