Direct observation of microtubules with the scanning tunneling microscope

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.